Anti-c1s antibodies and methods of use thereof

ABSTRACT

The present disclosure provides antibodies that specifically bind complement pathway component C1s. The present disclosure provides nucleic acids comprising nucleotide sequences encoding the anti-C1s antibodies; and host cells comprising the nucleic acids. The present disclosure provides compositions comprising the anti-C1s antibodies. The present disclosure provides methods of use of the anti-C1s antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/407,390 filed Oct. 12, 2016, which is incorporated herein byreference in its entirety.

REFERENCE To A SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web, and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 11, 2017, isnamed 4159_504PC01_SeqListing_ST25.TXT and is 72,260 Bytes in size.

BACKGROUND

The complement system is a well-known effector mechanism of the immuneresponse, providing not only protection against pathogens and otherharmful agents but also recovery from injury. The complement pathwaycomprises a number of proteins that typically exist in the body in aninactive form. The classical complement pathway is triggered byactivation of the first component of complement, referred to as the C1complex, which consists of C1q, C1r, and C1s proteins. Upon binding ofC1 to an immune complex or other activator, the C1s component, adiisopropyl fluorophosphate (DFP)-sensitive serine protease, cleavescomplement components C4 and C2 to initiate activation of the classicalcomplement pathway. The classical complement pathway appears to play arole in many diseases and disorders.

SUMMARY

The present disclosure provides humanized anti-C1s antibodies. Thepresent disclosure provides nucleic acids comprising nucleotidesequences encoding the humanized anti-C1s antibodies; and host cellscomprising the nucleic acids. The present disclosure providescompositions comprising the humanized anti-C1s antibodies. The presentdisclosure provides methods of use of the humanized anti-C1s antibodies.

Some aspects of the present disclosure are directed to an antibody,comprising a heavy chain and a light chain, wherein the heavy chaincomprises a heavy chain variable (VH) region and a heavy chain constantregion, and the light chain comprises a light chain variable (VL)region; wherein the VL region comprises a VL complementary determiningregion (CDR) 1, a VL CDR2, and a VL CDR3, and wherein the VH regioncomprises a VH CDR1, a VH CDR2, and a VH CDR3; wherein the VL CDR1comprises SEQ ID NO: 1; wherein the VL CDR2 comprises SEQ ID NO: 2;wherein the VL CDR3 comprises SEQ ID NO: 3; wherein the VH CDR1comprises SEQ ID NO: 4; wherein the VH CDR2 comprises SEQ ID NO: 5;wherein the VH CDR3 comprises SEQ ID NO: 6; wherein the heavy chainconstant region comprises an IgG4 constant region, wherein amino acidresidue 308 of the heavy chain constant region corresponding to SEQ IDNO: 28 is Leu, and amino acid residue 314 of the heavy chain constantregion corresponding to SEQ ID NO: 28 is Ser; and wherein the antibodyspecifically binds activated C1s.

Certain aspects of the present disclosure are directed to an antibody,comprising a heavy chain and a light chain, wherein the heavy chaincomprises a VH region and a heavy chain constant region, and the lightchain comprises a VL region; wherein the VL region comprises a VL CDR1,a VL CDR2, and a VL CDR3, and wherein the VH region comprises a VH CDR1,a VH CDR2, and a VH CDR3; wherein the VL CDR1 comprises SEQ ID NO: 1;wherein the VL CDR2 comprises SEQ ID NO: 2; wherein the VL CDR3comprises SEQ ID NO: 3; wherein the VH CDR1 comprises SEQ ID NO: 4;wherein the VH CDR2 comprises SEQ ID NO: 5; wherein the VH CDR3comprises SEQ ID NO: 6; wherein the heavy chain constant regioncomprises SEQ ID NO: 28; and wherein the antibody specifically bindsactivated C1s.

Other aspects of the present disclosure are directed to animmunoconjugate comprising an antibody disclosed herein.

Other aspects of the present disclosure are directed to a nucleotide ora set of nucleotides encoding an antibody disclosed herein.

Other aspects of the present disclosure are directed to methods ofinhibiting a complement pathway in a subject in need thereof, comprisingadministering to the subject a pharmaceutically effective amount of anantibody, immunoconjugate, or nucleotide disclosed herein.

Other aspects of the present disclosure are directed to methods oftreating a complement-mediated disease or disorder in a subject in needthereof, comprising administering to the subject a pharmaceuticallyeffective amount of an antibody, immunoconjugate, or nucleotidedisclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an amino acid sequence of humanized VH variant 1 (SEQ IDNO:10) and a nucleotide sequence (SEQ ID NO:11) encoding same. CDRdefinitions and protein sequence numbering are shown according to Kabatnumbering. CDR nucleotides and protein sequences are underlined.

FIG. 2 depicts an amino acid sequence of humanized VH variant 2 (SEQ IDNO:12) and a nucleotide sequence (SEQ ID NO:13) encoding same. CDRdefinitions and protein sequence numbering are shown according to Kabatnumbering. CDR nucleotides and protein sequences are underlined.

FIG. 3 depicts an amino acid sequence of humanized VH variant 3 (SEQ IDNO:14) and a nucleotide sequence (SEQ ID NO:15) encoding same. CDRdefinitions and protein sequence numbering are shown according to Kabatnumbering. CDR nucleotides and protein sequences are underlined.

FIG. 4 depicts an amino acid sequence of humanized VH variant 4 (SEQ IDNO:16) and a nucleotide sequence (SEQ ID NO:17) encoding same. CDRdefinitions and protein sequence numbering are shown according to Kabatnumbering. CDR nucleotides and protein sequences are underlined.

FIG. 5 depicts an amino acid sequence of humanized VH variant 5 (SEQ IDNO:18) and a nucleotide sequence (SEQ ID NO:19) encoding same. CDRdefinitions and protein sequence numbering are shown according to Kabatnumbering. CDR nucleotides and protein sequences are underlined.

FIG. 6 depicts an amino acid sequence of humanized Vκ variant 1 (SEQ IDNO:20) and a nucleotide sequence (SEQ ID NO:21) encoding same. CDRdefinitions and protein sequence numbering are shown according to Kabatnumbering. CDR nucleotides and protein sequences are underlined.

FIG. 7 depicts an amino acid sequence of humanized Vκ variant 2 (SEQ IDNO:22) and a nucleotide sequence (SEQ ID NO:23) encoding same. CDRdefinitions and protein sequence numbering are shown according to Kabatnumbering. CDR nucleotides and protein sequences are underlined.

FIG. 8 depicts an amino acid sequence of humanized Vκ variant 5 (SEQ IDNO:24) and a nucleotide sequence (SEQ ID NO:25) encoding same. CDRdefinitions and protein sequence numbering are shown according to Kabatnumbering. CDR nucleotides and protein sequences are underlined.

FIG. 9 shows amino acid differences between parental murineanti-activated C1s (anti-aC1s; also known as TNT005) VH (SEQ ID NO: 8)and exemplary humanized VH variants.

FIG. 10 shows amino acid differences between parental murine anti-aC1sVL (SEQ ID NO: 7) and exemplary humanized VL variants.

FIG. 11 shows binding properties of humanized variants of murineanti-aC1s. Data for direct binding to activated C1s (“aC1s”),competition binding with 50 pM biotinylated-anti-aC1s (“Biot-005”), andinhibition of the classical complement pathway, are shown.

FIG. 12 shows binding properties of humanized variants of murineanti-aC1s. Affinity data for binding of humanized variants of murineanti-aC1s are provided.

FIG. 13 depicts a pharmacokinetic (PK) profile and a pharmacodynamic(PD) profile for a humanized anti-aC1s variant (VH3/VK2-Fc-sub₄; alsoknown as TNT020) delivered intravenously at 10 mg/kg to cynomolgusmonkeys. The data show % complement pathway (CP) activity (normalized topre-administration level), and serum concentration (μg/mL) ofadministered antibody, at times up to 650 hours post-administration.

FIG. 14 depicts a PK profile and a PD profile for VH3/VK2-Fc-sub₄delivered subcutaneously at 20 mg/kg to cynomolgus monkeys. The datashow % CP activity (normalized to pre-administration level), and serumconcentration (μg/mL) of administered antibody, at times up to 55 dayspost-administration. The pharmacokinetic (circles) and pharmacodynamic(squares) profiles are overlaid. CP=complement pathway.

FIGS. 15A-15C provide amino acid sequences of a VH3/VK2-Fc-sub₄. FIG.15A provides the Fc-sub₄ amino acid sequence present in VH3/VK2-Fc-sub₄.Amino acid substitutions that enhance FcRn binding are underlined (FIG.15A). FIGS. 15B and 15C provide the heavy chain (FIG. 15B) and the lightchain (FIG. 15C) amino acid sequences of VH3/VK2-Fc-sub₄. Variableregion CDRs are underlined (FIGS. 15B and 15C), and the heavy chainconstant region (Fc domain) is bolded (FIG. 15B).

FIGS. 16A-16B provide amino acid sequences of the full length heavy andlight chains of VH3/VK2-Fc-sub₄. The signal peptides are bolded andunderlined (FIGS. 16A and 16B); the CDRs are underlined (FIGS. 16A and16B); and the heavy chain constant region (Fc) is bolded (FIG. 16A).Heavy chain constant region amino acid substitutions that enhance FcRnbinding are double-underlined (FIG. 16A).

FIGS. 17A-17B are graphical representations illustrating the serumcomplement pathway (CP) activity (FIG. 17A) and hemolysis (FIG. 17B) invitro, following exposure to varying concentrations of an anti-C1santibody that targets both active and inactive C1s (squares) orVH3/VK2-Fc-sub₄ (circles).

FIGS. 18A-18B are graphical representations illustrating thepharmacokinetic (FIG. 18A) and pharmacodynamic (FIG. 18B) profiles foran anti-aC1s antibody variant (VH3/VK2 having a wild-type IgG4 Fc) andVH3/VK2-Fc-sub₄ (having a heavy chain sequence comprising SEQ ID NO:28). A negative “vehicle” control is also shown in FIG. 18B.

DEFINITIONS

The term “complement component C1s” or “C1s,” as used herein, refers toa diisopropyl fluorophosphate (DFP)-sensitive serine protease, whichcleaves complement components C4 and C2 to initiate activation of theclassical complement pathway. The wild-type amino acid sequence forhuman C1s is provided in Table 1 (SEQ ID NO: 9).

TABLE 1 Sequences Human C1sEPTMYGEILSPNYPQAYPSEVEKSWDIEVPEGYGIHLYFTHLDIELSENCAYDSVQIISGDTEEGRLCGQRSSNNPHSPIVEEFQVPYNKLQVIFKSDFSNEERFTGFAAYYVATDINECTDFVDVPCSHFCNNFIGGYFCSCPPEYFLHDDMKNCGVNCSGDVFTALIGEIASPNYPKPYPENSRCEYQIRLEKGFQVVVTLRREDFDVEAADSAGNCLDSLVFVAGDRQFGPYCGHGFPGPLNIETKSNALDIIFQTDLTGQKKGWKLRYHGDPMPCPKEDTPNSVWEPAKAKYVERDVVQITCLDGFEVVEGRVGATSFYSTCQSNGKWSNSKLKCQPVDCGIPESIENGKVEDPESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVLGPELPKCVPVCGVPREPFEEKQRIIGGSDADIKNFPWQVFFDNPWAGGALINEYWVLTAAHVVEGNREPTMYVGSTSVQTSRLAKSKMLTPEHVFIHPGWKLLEVPEGRTNFDNDIALVRLKDPVKMGPTVSPICLPGTSSDYNLMDGDLGLISGWGRTEKRDRAVRLKAARLPVAPLRKCKEVKVEKPTADAEAYVFTPNMICAGGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCGTYGLYTRVKNYVDWIMKTMQENSTPRED (SEQ ID NO: 9) Human IgG4ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG ConstantLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLRegion (Fc)FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 52) Human IgG4ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG ConstantLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLRegion (Fc)FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV Variant 1SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS (S241P;LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC L248E)SVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 53) Human IgG4ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG ConstantLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLRegion (Fc)FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV Variant 2SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS (S241P;LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC L248E;SVLHEALHSHYTQKSLSLSLGK (SEQ ID NO: 28) M428L; N434S)

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, that retain specific binding to antigen.An “antibody” includes, without limitation, a glycoproteinimmunoglobulin which binds specifically to an antigen and comprises atleast two heavy (H) chains and two light (L) chains interconnected bydisulfide bonds, or an antigen-binding portion thereof. Each H chaincomprises a heavy chain variable region (abbreviated herein as V_(H))and a heavy chain constant region. The heavy chain constant regioncomprises three constant domains, C_(H1), C_(H2) and C_(H3). Each lightchain comprises a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion comprises one constant domain, C_(L). The V_(H) and V_(L) regionscan be further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) comprises three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. The variable regions of the heavy and light chainscontain a binding domain that interacts with an antigen. The constantregions of the antibodies can mediate the binding of the immunoglobulinto host tissues or factors, including various cells of the immune system(e.g., effector cells) and the first component (C1q) of the classicalcomplement system.

The term “antibody” includes, by way of example, both naturallyoccurring and non-naturally occurring antibodies; monoclonal andpolyclonal antibodies; chimeric and humanized antibodies; human ornonhuman antibodies; wholly synthetic antibodies; and single chainantibodies. A nonhuman antibody can be humanized by recombinant methodsto reduce its immunogenicity in man. Where not expressly stated, andunless the context indicates otherwise, the term “antibody” alsoincludes an antigen-binding fragment or an antigen-binding portion ofany of the aforementioned immunoglobulins, and includes a monovalent anda divalent fragment or portion, and a single chain antibody. Anantigen-binding fragment of an antibody can include any portion of anantibody that retains the ability to bind the target of the antibody. Insome embodiments, an antigen-binding fragment of an anti-C1s antibodyretains the ability to bind C1s. In some embodiments, an antigen-bindingfragment of an antibody comprises 1, 2, 3, 4, 5, or 6 CDRs of theantibody. In some embodiments, an antigen-binding fragment of anantibody comprises 1, 2, 3, 4, 5, or 6 CDRs and 1, 2, 3, 4, 5, 6, 7, or8 framework regions of the antibody. In some embodiments, anantigen-binding fragment of an antibody comprises a VH region and/or aVL region of the antibody.

The antibodies can be detectably labeled, e.g., with a radioisotope, anenzyme that generates a detectable product, a fluorescent protein, andthe like. The antibodies can be further conjugated to other moieties,such as members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), and the like. The antibodies canalso be bound to a solid support, including, but not limited to,polystyrene plates or beads, and the like. Also encompassed by the termare monoclonal antibodies. As used herein, a monoclonal antibody is anantibody produced by a group of identical cells, all of which wereproduced from a single cell by repetitive cellular replication. That is,the clone of cells only produces a single antibody species. While amonoclonal antibody can be produced using hybridoma productiontechnology, other production methods known to those skilled in the artcan also be used (e.g., antibodies derived from antibody phage displaylibraries). An antibody can be monovalent or bivalent. An antibody canbe an Ig monomer, which is a “Y-shaped” molecule that consists of fourpolypeptide chains: two heavy chains and two light chains connected bydisulfide bonds.

The term “monoclonal antibody” (“mAb”) refers to a non-naturallyoccurring antibody molecule of single molecular composition, i.e.,antibody molecules whose primary sequences are essentially identical,and which exhibits a single binding specificity and affinity for aparticular epitope. A mAb is an example of an isolated antibody. Theterm “monoclonal antibody” is not limited to antibodies prepared usinghybridoma techniques. Rather, monoclonal antibodies, as used herein, canbe produced by hybridoma, recombinant, transgenic or other techniquesknown to those skilled in the art.

The term “humanized immunoglobulin” or “humanized antibody” as usedherein refers to an immunoglobulin comprising portions ofimmunoglobulins of different origin, wherein at least one portioncomprises amino acid sequences of human origin. For example, thehumanized antibody can comprise portions derived from an immunoglobulinof nonhuman origin with the requisite specificity, such as a mouse, andfrom immunoglobulin sequences of human origin (e.g., chimericimmunoglobulin), joined together chemically by conventional techniques(e.g., synthetic) or prepared as a contiguous polypeptide using geneticengineering techniques (e.g., DNA encoding the protein portions of thechimeric antibody can be expressed to produce a contiguous polypeptidechain). Another example of a humanized immunoglobulin is animmunoglobulin containing one or more immunoglobulin chains comprising aCDR derived from an antibody of nonhuman origin and a framework regionderived from a light and/or heavy chain of human origin (e.g.,CDR-grafted antibodies with or without framework changes). In someembodiments, most or all of the amino acids outside the CDR regions of anon-human antibody are replaced with corresponding amino acids derivedfrom human immunoglobulins. In one embodiment of a humanized form of anantibody, some, most or all of the amino acids outside the CDR regionshave been replaced with amino acids from human immunoglobulins, whereassome, most or all amino acids within one or more CDR regions areunchanged. Chimeric or CDR-grafted single chain antibodies are alsoencompassed by the term humanized immunoglobulin. See, e.g., Cabilly etal., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No.0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533;Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S.Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, E.A. et al., European Patent Application No. 0,519,596 A1. See also,Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No. 5,476,786;and Bird, R. E. et al., Science, 242: 423-426 (1988)), regarding singlechain antibodies. Small additions, deletions, insertions, substitutionsor modifications of amino acids are permissible as long as they do notabrogate the ability of the antibody to bind to a particular antigen. Inparticular, conservative amino acid substitutions in one or moreframework region of the antibody are within the scope of the presentdisclosure. A “humanized” antibody retains an antigenic specificitysimilar to that of the original antibody.

For example, humanized immunoglobulins can be produced using syntheticand/or recombinant nucleic acids to prepare genes (e.g., cDNA) encodingthe desired humanized chain. For example, nucleic acid (e.g., DNA)sequences coding for humanized variable regions can be constructed usingPCR mutagenesis methods to alter DNA sequences encoding a human orhumanized chain, such as a DNA template from a previously humanizedvariable region (see e.g., Kamman, M., et al., Nucl. Acids Res., 17:5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993);Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991);and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)). Usingthese or other suitable methods, variants can also be readily produced.For example, cloned variable regions can be mutagenized, and sequencesencoding variants with the desired specificity can be selected (e.g.,from a phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548;Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)).

Humanization of a framework region(s) reduces the risk of the antibodyeliciting a human-anti-mouse-antibody (HAMA) response in humans.Art-recognized methods of determining immune response can be performedto monitor a HAMA response in a particular patient or during clinicaltrials. Patients administered humanized antibodies can be given animmunogenicity assessment at the beginning and throughout theadministration of the therapy. The HAMA response is measured, forexample, by detecting antibodies to the humanized therapeutic reagent,in serum samples from the patient using a method known to one in theart, including surface plasmon resonance technology (BIACORE) and/orsolid-phase enzyme-linked immunosorbent assay (ELISA) analysis. In manycases, a humanized anti-C1s antibody disclosed herein does notsubstantially elicit a HAMA response in a human subject.

Certain amino acids from the human variable region framework residuesare selected for substitution based on their possible influence on CDRconformation and/or binding antigen. The unnatural juxtaposition ofmurine CDR regions with human variable framework region can result inunnatural conformational restraints, which, unless corrected bysubstitution of certain amino acid residues, lead to loss of bindingaffinity.

A “chimeric antibody” refers to an antibody in which the variableregions are derived from one species and the constant regions arederived from another species, such as an antibody in which the variableregions are derived from a mouse antibody and the constant regions arederived from a human antibody.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains. Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses.

There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these classes can be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The subclassescan be further divided into types, e.g., IgG2a and IgG2b. “Isotype”refers to the antibody class or subclass (e.g., IgM or IgG1) that isencoded by the heavy chain constant region genes.

An “anti-antigen” antibody refers to an antibody that binds specificallyto the antigen. For example, an anti-C1s antibody binds specifically toC1s.

An “antigen-binding portion” of an antibody (also called an“antigen-binding fragment”) refers to one or more fragments of anantibody that retain the ability to bind specifically to the antigenbound by the whole antibody.

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents (e.g., an antibody and anantigen) and is expressed as a dissociation constant (K_(D)). Affinitycan be at least 1-fold greater, at least 2-fold greater, at least 3-foldgreater, at least 4-fold greater, at least 5-fold greater, at least6-fold greater, at least 7-fold greater, at least 8-fold greater, atleast 9-fold greater, at least 10-fold greater, at least 20-foldgreater, at least 30-fold greater, at least 40-fold greater, at least50-fold greater, at least 60-fold greater, at least 70-fold greater, atleast 80-fold greater, at least 90-fold greater, at least 100-foldgreater, or at least 1,000-fold greater, or more, than the affinity ofan antibody for unrelated amino acid sequences. Affinity of an antibodyto a target protein can be, for example, from about 100 nanomolar (nM)to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or fromabout 100 nM to about 1 femtomolar (fM) or more. As used herein, theterm “avidity” refers to the resistance of a complex of two or moreagents to dissociation after dilution. The terms “immunoreactive” and“preferentially binds” are used interchangeably herein with respect toantibodies and/or antigen-binding fragments.

The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, and ionicand/or hydrogen-bond interactions, including interactions such as saltbridges and water bridges. A humanized anti-C1s antibody of the presentdisclosure binds specifically to an epitope within a complement C1sprotein. “Specific binding” refers to binding with an affinity of atleast about 10⁻⁷ M or greater, e.g., 5×10⁻⁷ M, 10⁻⁸ M, 5×10⁻⁸ M, andgreater. “Non-specific binding” refers to binding with an affinity ofmore than about 10⁻⁷ M, e.g., binding with an affinity of 10⁻⁶ M, 10⁻⁵M, 10⁻⁴ M, etc. In some embodiments, the anti-C1s antibody specificallybinds to both the active and the inactive forms of C1s, e.g., withsimilar affinity. In certain embodiments, the anti-C1s antibodyspecifically binds to the active form of C1s and does not specificallybind to the inactive form of C1s.

As used herein, the term “CDR” or “complementarity determining region”is intended to mean the non-contiguous antigen combining sites foundwithin the variable region of both heavy and light chain polypeptides.CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616(1977); Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of proteins of immunological interest” (1991) (also referredto herein as Kabat 1991); by Chothia et al., J. Mol. Biol. 196:901-917(1987) (also referred to herein as Chothia 1987); and MacCallum et al.,J. Mol. Biol. 262:732-745 (1996), where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or grafted antibodies or variants thereof is intended tobe within the scope of the term as defined and used herein. The aminoacid residues, which encompass the CDRs, as defined by each of the abovecited references are set forth below in Table 2 as a comparison. TheCDRs depicted in FIGS. 1-8 were defined in accordance with Kabat 1991.

TABLE 2 CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H) CDR-1 31-3526-32 30-35 V_(H) CDR-2 50-65 53-55 47-58 V_(H) CDR-3 95-102 96-10193-101 V_(L) CDR-1 24-34 26-32 30-36 V_(L) CDR-2 50-56 50-52 46-55 V_(L)CDR-3 89-97 91-96 89-96 ¹Residue numbering follows the nomenclature ofKabat et al., supra ²Residue numbering follows the nomenclature ofChothia et al., supra ³Residue numbering follows the nomenclature ofMacCallum et al., supra

As used herein, the terms “CDR-L1,” “CDR-L2,” and “CDR-L3” refer,respectively, to the first, second, and third CDRs in a light chainvariable region. As used herein, the terms “CDR-H1,” “CDR-H2,” and“CDR-H3” refer, respectively, to the first, second, and third CDRs in aheavy chain variable region. As used herein, the terms “CDR-1,” “CDR-2,”and “CDR-3” refer, respectively, to the first, second and third CDRs ofeither chain's variable region.

As used herein, the term “framework” or “FR”, when used in reference toan antibody variable region, is intended to mean all amino acid residuesoutside the CDR regions within the variable region of an antibody. Avariable region framework is generally a discontinuous amino acidsequence between about 100-120 amino acids in length but is intended toreference only those amino acids outside of the CDRs. As used herein,the term “framework region” is intended to mean each domain of theframework that is separated by the CDRs. A light chain variable region(VL region) can have four framework regions: FR1, FR2, FR3, and FR4.Similarly, a heavy chain variable region (VH) can have four frameworkregions: FR1, FR2, FR3, and FR4.

The term “Fc domain” or “Fc region” as used herein, means functional FcR(e.g., FcRn) binding partners, unless otherwise specified. The Fc domainis the portion of a polypeptide which corresponds to the Fc domain ofnative Ig. A native Fc domain forms a homodimer with another Fc domain.In one embodiment, the “Fc region” refers to the portion of a single Igheavy chain beginning in the hinge region just upstream of the papaincleavage site (i.e. residue 216 in IgG, taking the first residue ofheavy chain constant region to be 114) and ending at the C-terminus ofthe antibody. In some embodiments, a complete Fc domain comprises atleast a hinge domain, a CH2 domain, and a CH3 domain. The Fc region ofan Ig constant region, depending on the Ig isotype can include the CH2,CH3, and CH4 domains, as well as the hinge region. In certainembodiments, the Fc region comprises SEQ ID NO: 52 (Table 3). In certainembodiments, the Fc region comprises SEQ ID NO: 53 (Table 3). In certainembodiments, the Fc region comprises SEQ ID NO: 28 (Table 3).

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and can include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, the antibody will bepurified (1) to greater than 90%, greater than 95%, or greater than 98%,by weight of antibody as determined by the Lowry method, for example,more than 99% by weight, (2) to a degree sufficient to obtain at least15 residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing ornonreducing conditions using Coomassie blue or silver stain. Isolatedantibody includes the antibody in situ within recombinant cells since atleast one component of the antibody's natural environment will not bepresent. In some instances, isolated antibody will be prepared by atleast one purification step.

The terms “polypeptide,” “peptide,” and “protein,” used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The term includes fusionproteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, with or without N-terminal methionineresidues; immunologically tagged proteins; and the like. A polypeptide,peptide, or protein can be naturally occurring or recombinant.

As used herein, the terms “treatment,” “treating,” “treat,” and thelike, refer to obtaining a desired pharmacologic and/or physiologiceffect. The effect can be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or can betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease, e.g., reducing orameliorating one or more symptoms of the disease. “Treatment,” as usedherein, covers any treatment of a disease in a mammal, particularly in ahuman, and includes: (a) preventing the disease from occurring in asubject which can be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; and (c) relieving the disease, i.e., causing regression ofthe disease.

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein, refer to a mammal, including, but not limitedto, murines (rats, mice), non-human primates, humans, canines, felines,ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.Also encompassed by these terms are any animal that has a complementsystem, such as mammals, fish, and some invertebrates. As such theseterms include complement system-containing mammal, fish, andinvertebrate companion animals, agricultural animals, work animals, zooanimals, and lab animals.

A “therapeutically effective amount,” “pharmaceutically effectiveamount,” “effective amount,” or “efficacious amount” refers to theamount of an anti-complement C1s antibody that, when administered to amammal or other subject for treating a disease, is sufficient to effectsuch treatment for the disease. The “therapeutically effective amount”will vary depending on the anti-complement C1s antibody, the disease andits severity and the age, weight, etc., of the subject to be treated.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such aspolynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples. The term“biological sample” includes urine, saliva, cerebrospinal fluid,interstitial fluid, ocular fluid, synovial fluid, blood fractions suchas plasma and serum, and the like. The term “biological sample” alsoincludes solid tissue samples, tissue culture samples, and cellularsamples.

The term “substitution,” as used herein, refers to a difference betweena given sequence and a reference sequence. “Substitution” is not limitedto a particular method of arriving at the recited sequence.“Substitution” can be contrasted with “deleted,” which indicates thatone or more amino acids or nucleotides are missing in a given sequencerelative to a reference sequence. In both cases, a given sequence can besaid to have a substituted or deleted amino acid or nucleotideregardless of the origin of the sequence. For example, a given sequencecan be said to have a substitution at position 100 relative to areference sequence, even though the given sequence was created de novo,e.g., synthetically, and not through mutation of the reference sequence.In some embodiments, a substitution can comprise more than one aminoacid replacing a single amino acid.

The terms “cross competes” and “cross competition,” as used herein,refer to the ability of an antibody to compete for binding to a targetantigen with a reference antibody. Any methods known in the art can beused to determine whether an antibody cross competes with a referenceantibody. For example, BIAcore analysis, ELISA assays, or flow cytometrycan be used to demonstrate cross-competition with the antibodies of thecurrent disclosure. The ability of a test antibody to inhibit thebinding of an antibody to human C1s demonstrates that the test antibodycan compete with a reference antibody for binding to human C1s. In someembodiments, an antibody that cross competes with a reference antibodyfor binding to an antigen, e.g., human C1s, binds the same epitope asthe reference antibody. In some embodiments, an antibody that crosscompetes with a reference antibody for binding to an antigen, e.g.,human C1s, binds an epitope that is near or adjacent to the epitoperecognized by the reference antibody. In some embodiments, an antibodythat cross competes with a reference antibody for binding to an antigen,e.g., human C1s, binds an epitope that is distal to the epitoperecognized by the reference antibody; however, binding of the antibodyto the distal epitope is sufficient to disrupt the binding ability ofthe reference antibody to the antigen. An antibody binds the sameepitope as a reference antibody if the antibody interacts with aminoacid residues on the antigen which are the same as or overlap with theamino acids on the antigen that interact with the reference antibody.

Before the present disclosure is further described, it is to beunderstood that this disclosure is not limited to particular embodimentsdescribed, as such can, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present disclosure will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges can independently be included in thesmaller ranges, and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure. Inaddition, the term “about” is used herein to mean approximately,roughly, around, or in the regions of. When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Thus,“about 10-20” means “about 10 to about 20.” In general, the term “about”can modify a numerical value above and below the stated value by avariance of, e.g., 10 percent, up or down (higher or lower).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. All publications mentioned herein are incorporated herein byreference to disclose and describe the methods and/or materials inconnection with which the publications are cited.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects ofthe disclosure, which can be had by reference to the specification as awhole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahumanized anti-C1s antibody” includes a plurality of such antibodies andreference to “the framework region” includes reference to one or moreframework regions and equivalents thereof known to those skilled in theart, and so forth. It is further noted that the claims can be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

It is appreciated that certain features of the disclosure, which are,for clarity, described in the context of separate embodiments, can alsobe provided in combination in a single embodiment. Conversely, variousfeatures of the disclosure, which are, for brevity, described in thecontext of a single embodiment, can also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the disclosure are specifically embraced by the presentdisclosure and are disclosed herein just as if each and everycombination was individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present disclosure and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present disclosure isnot entitled to antedate such publication by virtue of prior disclosure.Further, the dates of publication provided can be different from theactual publication dates which can need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides an antibody, e.g., a humanized antibody,that binds complement C1s protein (i.e., an anti-complement C1santibody, also referred to herein as a “anti-C1s antibody,” a “C1santibody,” and a “subject antibody”) and a nucleic acid comprising anucleotide sequence that encodes such an antibody. In some aspects, theanti-C1s antibody specifically binds active C1s. In certain embodiments,the anti-C1s antibody does not specifically bind inactive C1s. In someaspects, the anti-C1s antibody is a humanized antibody. In otheraspects, the anti-C1s antibody of the present disclosure has one or moreimproved pharmacokinetic properties, e.g., improved half-life,stability, etc. In certain aspects, the anti-C1s antibody of the presentdisclosure can be administered subcutaneously. The present disclosurealso provides a composition comprising an antibody, e.g., a humanizedanti-C1s antibody, of the present disclosure. The present disclosureprovides methods of producing and using antibodies, nucleic acids, andcompositions of the present disclosure. The present disclosure providesmethods of treating a complement-mediated disease or disorder, involvingadministering an antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure.

Anti-Complement C1s Antibodies

The present disclosure provides anti-complement C1s antibodies, e.g.,humanized anti-complement C1s antibodies, and pharmaceuticalcompositions comprising such antibodies. Complement C1s is an attractivetarget as it is upstream in the complement cascade and has a narrowrange of substrate specificity. Of interest in some cases is an antibodythat specifically binds the activated form of C1s, e.g., where theantibody does not substantially bind the inactive form of C1s.

The present disclosure provides an anti-complement C1s antibody, e.g., ahumanized anti-complement C1s antibody, comprising:

a) a heavy chain comprising: i) a VH region comprising the amino acidsequence:(Q/E)VQL(V/Q)QSGAE(V/L)KKPGASVK(L/V)SC(T/A)ASGFNIKDDYIHWV(K/R)QAPGQGLEWIGRIDPADGHTKYAPKFQVK(V/A)TITADTST(S/N)TAY(L/M)(E/Q)LS SL(R/T) SEDTAVYYCARYGYGREVFDYWGQGTTVTVSS (SEQ ID NO:26); and ii) an Fc regioncomprising an amino acid sequence having at least 98% amino acidsequence identity with the amino acid sequence set forth in SEQ IDNO:28, wherein amino acid 308 is Leu and amino acid 314 is Ser; andb) a light chain comprising: i) a VL region comprising the amino acidsequence:DIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQK(T/P)GQPPK(I/L)LIYDASNLESGIPARF SGSGSGTDFTLTIS SLE(E/P)EDFA(I/V)YYCQQ SNEDPWTFGGGTKVEIK (SEQID NO:27); and ii) a light chain constant region.

In certain aspects of the present disclosure, the anti-C1s antibodycomprises a heavy chain constant region, comprising an Fc region. Insome embodiments, the heavy chain constant region comprises animmunoglobulin constant region, e.g., a human IgG constant region (e.g.,IgG Fc), or a variant thereof. In some embodiments, the heavy chainconstant region of the anti-C1s antibody is derived from a humanimmunoglobulin. In some embodiments, heavy chain constant region of theanti-C1s antibody comprises an IgG4 Fc or a variant thereof.

In some embodiments, the heavy chain Fc of the anti-C1s antibodycomprises an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% sequenceidentity with human IgG4 Fc (SEQ ID NO: 52) or SEQ ID NO: 28, whereinamino acid 308 is Leu and amino acid 314 is Ser. In some embodiments,the heavy chain Fc of the anti-C1s antibody comprises an amino acidsequence having at least about 98% sequence identity with human IgG4 Fc(SEQ ID NO: 52) or SEQ ID NO: 28, wherein amino acid 308 is Leu andamino acid 314 is Ser. In some aspects, the Fc region comprises an aminoacid sequence at least 99% identical to SEQ ID NO: 52 or SEQ ID NO: 28,wherein amino acid 308 is Leu and amino acid 314 is Ser. In otheraspects, the Fc region for an anti-C1s antibody comprises, consistsessentially of, or consists of SEQ ID NO: 28.

In certain aspects, an anti-C1s antibody of the present disclosure hasone or more improved pharmacokinetics, e.g., longer half-life,stability, etc, compared to wild type Fc region (SEQ ID NO: 52). Incertain embodiments, the anti-C1s antibodies comprising a heavy chain Fcregion comprising SEQ ID NO: 28 have a longer half-life than comparableantibodies having a wild type Fc region, e.g., a human IgG4 Fc. In otherembodiments, the anti-C1s antibodies are more stable followingsubcutaneous administration than comparable antibodies having a wildtype Fc region, e.g., a human IgG4 Fc. Accordingly, in some aspects, ananti-C1s antibody of the present disclosure can be administeredsubcutaneously.

In some embodiments, the heavy chain Fc of the anti-C1s antibodycomprises a substitution relative to human IgG4 Fc. In some embodiments,the anti-C1s antibody comprises an Fc having at least about 98% sequenceidentity to human IgG4 Fc. In certain embodiments, the anti-C1s antibodycomprises an Fc, wherein the amino acid residue corresponding to aminoacid 108 of SEQ ID NO: 28, when properly aligned, is a proline (e.g., aS241P variant of human IgG4). In certain embodiments, the anti-C1santibody comprises an Fc, wherein the amino acid residue correspondingto amino acid 115 of SEQ ID NO: 28, when properly aligned, is a glutamicacid (e.g., a L248E variant of human IgG4). In certain embodiments, theanti-C1s antibody comprises an Fc, wherein the amino acid residuecorresponding to amino acid 308 of SEQ ID NO: 28, when properly aligned,is a leucine (e.g., a M428L variant of human IgG4). In certainembodiments, the anti-C1s antibody comprises an Fc, wherein the aminoacid residue corresponding to amino acid 314 of SEQ ID NO: 28, whenproperly aligned, is a serine (e.g., a N434S variant of human IgG4).

In some embodiments, the anti-C1s antibody comprises an Fc, wherein: (a)the amino acid residue corresponding to amino acid 108 of SEQ ID NO: 28,when properly aligned, is a proline; (b) the amino acid residuecorresponding to amino acid 115 of SEQ ID NO: 28, when properly aligned,is a glutamic acid; (c) the amino acid residue corresponding to aminoacid 308 of SEQ ID NO: 28, when properly aligned, is a leucine; (d) theamino acid residue corresponding to amino acid 314 of SEQ ID NO: 28,when properly aligned, is a serine; (e) or any combination of (a), (b),(c), and (d). In some embodiments, the anti-C1s antibody comprises anFc, wherein: (a) the amino acid residue corresponding to amino acid 108of SEQ ID NO: 28, when properly aligned, is a proline; (b) the aminoacid residue corresponding to amino acid 115 of SEQ ID NO: 28, whenproperly aligned, is a glutamic acid; (c) the amino acid residuecorresponding to amino acid 308 of SEQ ID NO: 28, when properly aligned,is a leucine; and (d) the amino acid residue corresponding to amino acid314 of SEQ ID NO: 28, when properly aligned, is a serine.

In some embodiments, the Fc of the anti-C1s antibody has a greaterbinding affinity to an Fc receptor, e.g., FcRn, than human IgG4.

In some embodiments, the anti-C1s antibody, e.g., the humanized anti-C1santibody, comprises an Fc comprising the amino acid sequence set forthin SEQ ID NO:28. In some embodiments, the anti-C1s antibody, e.g., thehumanized anti-C1s antibody, comprises: a) an Fc comprising the aminoacid sequence set forth in SEQ ID NO:28; and b) a light chain comprisinga human Vκ constant region.

Variable Regions

In some embodiments, an antibody of the present disclosure comprises aheavy chain and a light chain, wherein the heavy chain comprises a heavychain variable (VH) region and a heavy chain constant region, and thelight chain comprises a light chain variable (VL) region; wherein the VLregion comprises a VL complementary determining region (CDR) 1comprising SEQ ID NO: 1, a VL CDR2 comprising SEQ ID NO: 2, and a VLCDR3 comprising SEQ ID NO: 3, and wherein the VH region comprises a VHCDR1 comprising SEQ ID NO: 4, a VH CDR2 comprising SEQ ID NO: 5, and aVH CDR3 comprising SEQ ID NO: 6; wherein the heavy chain constant regioncomprises an IgG4 constant region, wherein amino acid residue 308 of theheavy chain constant region corresponding to SEQ ID NO: 28 is a leucine,and amino acid residue 314 of the heavy chain constant regioncorresponding to SEQ ID NO: 28 is a serine; and wherein the antibodyspecifically binds activated C1s. In some embodiments, amino acidresidue 108 of the heavy chain constant region corresponding to SEQ IDNO: 28 is a proline. In some embodiments, amino acid residue 115 of theheavy chain constant region corresponding to SEQ ID NO: 28 is a glutamicacid.

In some embodiments, an antibody of the present disclosure comprises aheavy chain and a light chain, wherein the heavy chain comprises a VHregion and a heavy chain constant region, and the light chain comprisesa VL region; wherein the VL region comprises a VL CDR1 comprising SEQ IDNO: 1, a VL CDR2 comprising SEQ ID NO: 2, and a VL CDR3 comprising SEQID NO: 3, and wherein the VH region comprises a VH CDR1 comprising SEQID NO: 4, a VH CDR2 comprising SEQ ID NO: 5, and a VH CDR3 comprisingSEQ ID NO: 6; wherein the heavy chain constant region comprises SEQ IDNO: 28; and wherein the antibody specifically binds activated C1s.

In some embodiments, the antibody is a human antibody. In someembodiments, the antibody is a humanized antibody. In some embodiments,the antibody is a chimeric antibody. In some embodiments, the antibodyis a murine antibody.

In some embodiments, the antibody is a monoclonal antibody. In someembodiments, the antibody is a recombinant antibody. In someembodiments, the antibody is a synthetic antibody.

In some cases, an anti-C1s antibody of the present disclosure inhibitsC1s-mediated cleavage of complement component C4. In some embodiments,the anti-C1s antibody inhibits enzymatic activity of the serine-proteasedomain of C1s. In some cases, an anti-C1s antibody of the presentdisclosure inhibits C1s-mediated cleavage of complement component C2. Insome cases, an anti-C1s antibody of the present disclosure inhibitsC1s-mediated cleavage of C4 and C2. In some embodiments, the anti-C1santibody of the present disclosure depletes C1s/aC1s from circulation.

In some embodiments, the anti-C1s antibody is a humanized antibody. Insome cases, a humanized anti-C1s antibody of the present disclosureincludes at least one humanized V_(H) framework region. In some cases,an anti-C1s antibody of the present disclosure includes at least onehumanized V_(L) framework region. In some cases, an anti-C1s antibody ofthe present disclosure includes at least one humanized V_(H) frameworkregion and at least one humanized V_(L) framework region.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure includes VL CDRs present in thefollowing amino acid sequence:DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKTGQPPKILIYDASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAAIYYCQQSNEDPWTFGGGTKLEIK (SEQ ID NO: 7). Insome cases, a humanized anti-C1s antibody of the present disclosureincludes VH CDRs present in the following amino acid sequence:EVQLQQSGAELVRPGASVKLSCTASGFNIKDDYIHWVKQRPEQGLEWIGRIDPADGHTKYAPKFQVKATITADTSSNTAYLQLSSLTSEDTAVYYCARYGYGREVFDYWGQGTTLTVS S (SEQ IDNO: 8). In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody of the present disclosure includes VL CDRs present in SEQ IDNO: 7 and VH CDRs present in SEQ ID NO: 8.

In some embodiments, the anti-C1s antibody comprises one or more VL CDRspresent in SEQ ID NO: 22. In some embodiments, the anti-C1s antibodycomprises a VL CDR1 present in SEQ ID NO: 22. In some embodiments, theanti-C1s antibody comprises a VL CDR2 present in SEQ ID NO: 22. In someembodiments, the anti-C1s antibody comprises a VL CDR3 present in SEQ IDNO: 22. In some embodiments, the anti-C1s antibody comprises one or moreVH CDRs present in SEQ ID NO: 14. In some embodiments, the anti-C1santibody comprises a VH CDR1 present in SEQ ID NO: 14. In someembodiments, the anti-C1s antibody comprises a VH CDR2 present in SEQ IDNO: 14. In some embodiments, the anti-C1s antibody comprises a VH CDR3present in SEQ ID NO: 14.

In some embodiments, the anti-C1s antibody comprises one or more VL CDRspresent in SEQ ID NO: 30. In some embodiments, the anti-C1s antibodycomprises a VL CDR1 present in SEQ ID NO: 30. In some embodiments, theanti-C1s antibody comprises a VL CDR2 present in SEQ ID NO: 30. In someembodiments, the anti-C1s antibody comprises a VL CDR3 present in SEQ IDNO: 30. In some embodiments, the anti-C1s antibody comprises one or moreVH CDRs present in SEQ ID NO: 29. In some embodiments, the anti-C1santibody comprises a VH CDR1 present in SEQ ID NO: 29. In someembodiments, the anti-C1s antibody comprises a VH CDR2 present in SEQ IDNO: 29. In some embodiments, the anti-C1s antibody comprises a VH CDR3present in SEQ ID NO: 29.

In certain embodiments, the ant-C1s antibody comprises a VL CDR1(CDR-L1) comprising SEQ ID NO:1: KASQSVDYDGDSYMN. In some embodiments,the ant-C1s antibody comprises a VL CDR1 (CDR-L1) comprising SEQ ID NO:33: SQSVDYDGDSY. In some embodiments, the ant-C1s antibody comprises aVL CDR1 (CDR-L1) comprising SEQ ID NO: 39: DSYMNWY.

In certain embodiments, the ant-C1s antibody comprises a VL CDR2(CDR-L2) comprising SEQ ID NO:2: DASNLES. In some embodiments, theant-C1s antibody comprises a VL CDR2 (CDR-L2) comprising SEQ ID NO: 34:DAS. In some embodiments, the ant-C1s antibody comprises a VL CDR2(CDR-L2) comprising SEQ ID NO: 40: ILIYDASNLE.

In certain embodiments, the ant-C1s antibody comprises a VL CDR3(CDR-L3) comprising SEQ ID NO:3: QQSNEDPWT. In some embodiments, theant-C1s antibody comprises a VL CDR3 (CDR-L3) comprising SEQ ID NO: 35:SNEDPW. In some embodiments, the ant-C1s antibody comprises a VL CDR3(CDR-L3) comprising SEQ ID NO: 41: QQSNEDPW.

In certain embodiments, the ant-C1s antibody comprises a VH CDR1(CDR-H1) comprising SEQ ID NO:4: DDYIH. In some embodiments, the ant-C1santibody comprises a VH CDR1 (CDR-H1) comprising SEQ ID NO: 36: GFNIKDD.In some embodiments, the ant-C1s antibody comprises a VH CDR1 (CDR-H1)comprising SEQ ID NO: 42: KDDYIH.

In certain embodiments, the ant-C1s antibody comprises a VH CDR2(CDR-H2) comprising SEQ ID NO:5: RIDPADGHTKYAPKFQV. In some embodiments,the anti-C1s antibody comprises a VH CDR2 (CDR-H2) comprising SEQ ID NO:37: ADG. In some embodiments, the anti-C1s antibody comprises a VH CDR2(CDR-H2) comprising SEQ ID NO: 43: WIGRIDPADGHTK.

In certain embodiments, the ant-C1s antibody comprises a VH CDR3(CDR-H3) comprising SEQ ID NO:6: YGYGREVFDY. In some embodiments, theanti-C1s antibody comprises a VH CDR3 (CDR-H3) comprising SEQ ID NO: 38:GYGREVFD. In some embodiments, the anti-C1s antibody comprises a VH CDR3(CDR-H3) comprising SEQ ID NO: 44: ARYGYGREVFD.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a light chain variableregion comprising CDR amino acid sequences SEQ ID NO:1, SEQ ID NO:2, andSEQ ID NO:3 (CDR-L1, CDR-L2, and CDR-L3, respectively).

In some embodiments, an anti-C1s antibody of the present disclosurecomprises a heavy chain variable region comprising CDR amino acidsequences SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 (CDR-H1, CDR-H2, andCDR-H3, respectively).

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VH region comprising thefollowing sequence:

(SEQ ID NO: 26) (Q/E)VQL(V/Q)QSGAE(V/L)KKPGASVK(L/V)SC(T/A)ASGFNIKDDYIHWV(K/R)QAPGQGLEWIGRIDPADGHTKYAPKFQVK(V/A)TITADTST(S/N)TAY(L/M)(E/Q)LSSL(R/T)SEDTAVYYCARYGYGREVFDYWGQG TTVTVSS.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VH region comprising anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of Table 3. In certain embodiments,the VH region of the antibody comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 10, 12, 14, 16, and 18 (Table3). In some embodiments, the VH region of the antibody comprises SEQ IDNO:14.

TABLE 3 Variable Heavy Chain Variants Variant Variable HeavyMature Heavy Chain Parental EVQLQQSGAELVRPGASVEVQLQQSGAELVRPGASVKLSCTASGFNIKDDYIHWVKQRPEQGLEWIG MurineKLSCTASGFNIKDDYIHW RIDPADGHTKYAPKFQVKATITADTSSNTAYLQLSSLTSEDTAVYYCARanti-C1s VKQRPEQGLEWIGRIDPAYGYGREVEDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VH DGHTKYAPKFQVKATITAVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG DTSSNTAYLQLSSLTSEDTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPK TAVYYCARYGYGREVFDYPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ WGQGTTLTVSS (SEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR ID NO: 8)EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVESCSVLHEALHSHYTQKSLS LSLGK (SEQ ID NO: 53)VH1 EVQLVQSGAELKKPGASV EVQLVQSGAELKKPGASVKLSCTASGFNIKDDYIHWVKQAPGQGLEWIGKLSCTASGFNIKDDYIHW RIDPADGHTKYAPKFQVKATITADTSTNTAYLQLSSLTSEDTAVYYCARVKQAPGQGLEWIGRIDPA YGYGREVFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLDGHTKYAPKFQVKATITA VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGDTSTNTAYLQLSSLTSED TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKTAVYYCARYGYGREVFDY PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQWGQGTTVTVSS (SEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRID NO: 10) EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLS LSLGK (SEQ ID NO: 46)VH2 EVQLVQSGAEVKKPGASV EVQLVQSGAEVKKPGASVKLSCTASGENIKDDYTHWVKQAPGQGLEWIGKLSCTASGENIKDDYTHW RIDPADGHTKYAPKFQVKATITADTSTNTAYLELSSLRSEDTAVYYCARVKQAPGQGLEWIGRIDPA YGYGREVFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLDGHTKYAPKFQVKATITA VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGDTSTNTAYLELSSLRSED TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKTAVYYCARYGYGREVFDY PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQWGQGTTVTVSS (SEQ ENSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRID NO: 12) EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLS LSLGK (SEQ ID NO: 47)VH3 QVQLVQSGAEVKKPGASV QVQLVQSGAEVKKPGASVKLSCTASGFNIKDDYIHWVKQAPGQGLEWIGKLSCTASGFNIKDDYIHW RIDPADGHTKYAPKFQVKVTITADTSTSTAYLELSSLRSEDTAVYYCARVKQAPGQGLEWIGRIDPA YGYGREVFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLDGHTKYAPKFQVKVTITA VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGDTSTSTAYLELSSLRSED TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKTAVYYCARYGYGREVFDY PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQWGQGTTVTVSS (SEQ ENSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRID NO: 14) EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLS LSLGK (SEQ ID NO: 29)VH4 QVQLVQSGAEVKKPGASV QVQLVQSGAEVKKPGASVKVSCTASGENIKDDYTHWVRQAPGQGLEWIGKVSCTASGENIKDDYTHW RIDPADGHTKYAPKFQVKVTITADTSTSTAYMELSSLRSEDTAVYYCARVRQAPGQGLEWIGRIDPA YGYGREVFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLDGHTKYAPKFQVKVTITA VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGDTSTSTAYMELSSLRSED TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKTAVYYCARYGYGREVFDY PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQWGQGTTVTVSS (SEQ ENSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRID NO: 16) EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLS LSLGK (SEQ ID NO: 48)VH5 QVQLVQSGAEVKKPGASV QVQLVQSGAEVKKPGASVKVSCAASGENIKDDYTHWVRQAPGQGLEWIGKVSCAASGENIKDDYTHW RIDPADGHTKYAPKFQVKVTITADTSTSTAYMELSSLRSEDTAVYYCARVRQAPGQGLEWIGRIDPA YGYGREVFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLDGHTKYAPKFQVKVTITA VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGDTSTSTAYMELSSLRSED TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKTAVYYCARYGYGREVFDY PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQWGQGTTVTVSS (SEQ ENSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRID NO: 18) EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLS LSLGK (SEQ ID NO: 49)

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VH region comprising anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 1, and set forth inSEQ ID NO:10, where amino acid 1 is Glu, amino acid 5 is Val, amino acid11 is Leu, amino acid 12 is Lys, amino acid 13 is Lys, amino acid 20 isLeu, amino acid 23 is Thr, amino acid 38 is Lys, amino acid 40 is Ala,amino acid 42 is Gly, amino acid 67 is Ala, amino acid 75 is Thr, aminoacid 76 is Asn, amino acid 80 is Leu, amino acid 81 is Gln, amino acid83 is Thr, and amino acid 109 is Val, where the numbering of the aminoacids is as depicted in FIG. 1.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VH region comprising theamino acid sequence depicted in FIG. 1, and set forth in SEQ ID NO:10.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VH region comprising anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2 and set forth inSEQ ID NO:12, where amino acid 1 is Glu, amino acid 5 is Val, amino acid11 is Val, amino acid 12 is Lys, amino acid 13 is Lys, amino acid 20 isLeu, amino acid 23 is Thr, amino acid 38 is Lys, amino acid 40 is Ala,amino acid 42 is Gly, amino acid 67 is Ala, amino acid 75 is Thr, aminoacid 76 is Asn, amino acid 80 is Leu, amino acid 81 is Glu, amino acid83 is Arg, and amino acid 109 is Val, where the numbering of the aminoacids is as depicted in FIG. 2.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VH region comprising theamino acid sequence depicted in FIG. 2, and set forth in SEQ ID NO:12.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VH region comprising anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 3 and set forth inSEQ ID NO:14, where amino acid 1 is Gln, amino acid 5 is Val, amino acid11 is Val, amino acid 12 is Lys, amino acid 13 is Lys, amino acid 20 isLeu, amino acid 23 is Thr, amino acid 38 is Lys, amino acid 40 is Ala,amino acid 42 is Gly, amino acid 67 is Val, amino acid 75 is Thr, aminoacid 76 is Ser, amino acid 80 is Leu, amino acid 81 is Glu, amino acid83 is Arg, and amino acid 109 is Val, where the numbering of the aminoacids is as depicted in FIG. 3.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VH region comprising theamino acid sequence depicted in FIG. 3, and set forth in SEQ ID NO:14.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VH region comprising anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 4 and set forth inSEQ ID NO:16, where amino acid 1 is Gln, amino acid 5 is Val, amino acid11 is Val, amino acid 12 is Lys, amino acid 13 is Lys, amino acid 20 isVal, amino acid 23 is Thr, amino acid 38 is Arg, amino acid 40 is Ala,amino acid 42 is Gly, amino acid 67 is Val, amino acid 75 is Thr, aminoacid 76 is Ser, amino acid 80 is Met, amino acid 81 is Glu, amino acid83 is Arg, and amino acid 109 is Val, where the numbering of the aminoacids is as depicted in FIG. 4.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VH region comprising theamino acid sequence depicted in FIG. 4, and set forth in SEQ ID NO:16.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VH region comprising anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 5 and set forth inSEQ ID NO:18, where amino acid 1 is Gln, amino acid 5 is Val, amino acid11 is Val, amino acid 12 is Lys, amino acid 13 is Lys, amino acid 20 isVal, amino acid 23 is Ala, amino acid 38 is Arg, amino acid 40 is Ala,amino acid 42 is Gly, amino acid 67 is Val, amino acid 75 is Thr, aminoacid 76 is Ser, amino acid 80 is Met, amino acid 81 is Glu, amino acid83 is Arg, and amino acid 109 is Val, where the numbering of the aminoacids is as depicted in FIG. 5.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VH region comprising theamino acid sequence depicted in FIG. 5, and set forth in SEQ ID NO:18.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VL region comprising thefollowing sequence:

(SEQ ID NO: 27) DIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQK(T/P)GQPPK(I/L)LIYDASNLESGIPARFSGSGSGTDFTLTISSLE(E/P)EDFA(I/V)YYCQQSNEDPWTFGGGTKVEIK.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VL region comprising anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of Table 4. In certain embodiments,the VL region of the antibody comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 20, 22, and 24 (Table 4). Insome embodiments, the VL region of the antibody comprises SEQ ID NO: 22.

TABLE 4 Variable Light Chain Variants Variant Variable LightMature Light Chain Parental DIVLTQSPASLAVSLGQRATISDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKTGQ MurineCKASQSVDYDGDSYMNWYQQKT PPKILIYDASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAAIYYCanti-C1s GQPPKILIYDASNLESGIPARFQQSNEDPWTEGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVC VL SGSGSGTDFTLNIHPVEEEDAALLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT IYYCQQSNEDPWTFGGGTKLEILSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: K (SEQ ID NO: 7) 54) VK1DIVLTQSPDSLAVSLGERATIS DIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQKTGQCKASQSVDYDGDSYMNWYQQKT PPKILIYDASNLESGIPARFSGSGSGTDFTLTISSLEEEDFAIYYCGQPPKILIYDASNLESGIPARF QQSNEDPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCSGSGSGTDFTLTISSLEEEDFA LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTIYYCQQSNEDPWTFGGGTKVEI LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:K (SEQ ID NO: 20) 50) VK2 DIVLTQSPDSLAVSLGERATISDIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQKPGQ CKASQSVDYDGDSYMNWYQQKPPPKILIYDASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAIYYC GQPPKILIYDASNLESGIPARFQQSNEDPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC SGSGSGTDFTLTISSLEPEDFALLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT IYYCQQSNEDPWTFGGGTKVEILSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: K (SEQ ID NO: 22) 30) VK5DIVLTQSPDSLAVSLGERATIS DIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQKPGQCKASQSVDYDGDSYMNWYQQKP PPKLLIYDASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCGQPPKLLIYDASNLESGIPARF QQSNEDPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCSGSGSGTDFTLTISSLEPEDFA LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTVYYCQQSNEDPWTFGGGTKVEI LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:K (SEQ ID NO: 24) 51)

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VL region comprising anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 6, and set forth inSEQ ID NO:20, where amino acid 9 is Asp, amino acid 17 is Glu, aminoacid 40 is Thr, amino acid 46 is Ile, amino acid 74 is Thr, amino acid76 is Ser, amino acid 77 is Ser, amino acid 78 is Leu, amino acid 80 isGlu, amino acid 83 is Phe, amino acid 85 is Ile, and amino acid 104 isVal, where the numbering of the amino acids is as depicted in FIG. 6.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VL region comprising theamino acid sequence depicted in FIG. 6, and set forth in SEQ ID NO:20.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VL region comprising anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 7, and set forth inSEQ ID NO:22, where amino acid 9 is Asp, amino acid 17 is Glu, aminoacid 40 is Pro, amino acid 46 is Ile, amino acid 74 is Thr, amino acid76 is Ser, amino acid 77 is Ser, amino acid 78 is Leu, amino acid 80 isPro, amino acid 83 is Phe, amino acid 85 is Ile, and amino acid 104 isVal, where the numbering of the amino acids is as depicted in FIG. 7.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VL region comprising theamino acid sequence depicted in FIG. 7, and set forth in SEQ ID NO:22.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VL region comprising anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 8 and set forth inSEQ ID NO:24, where amino acid 9 is Asp, amino acid 17 is Glu, aminoacid 40 is Pro, amino acid 46 is Leu, amino acid 74 is Thr, amino acid76 is Ser, amino acid 77 is Ser, amino acid 78 is Leu, amino acid 80 isPro, amino acid 83 is Phe, amino acid 85 is Val, and amino acid 104 isVal, where the numbering of the amino acids is as depicted in FIG. 8.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises a VL region comprising theamino acid sequence depicted in FIG. 8, and set forth in SEQ ID NO:24.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 1 aminoacid sequence depicted in FIG. 1 and as set forth in SEQ ID NO: 10; andb) a VL variant 1 amino acid sequence depicted in FIG. 6 and as setforth in SEQ ID NO: 20.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 1 aminoacid sequence depicted in FIG. 1 and as set forth in SEQ ID NO:10; andb) a VL variant 2 amino acid sequence depicted in FIG. 7 and as setforth in SEQ ID NO:22.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 1 aminoacid sequence depicted in FIG. 1 and as set forth in SEQ ID NO:10; andb) a VL variant 5 amino acid sequence depicted in FIG. 8 and as setforth in SEQ ID NO:24.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 2 aminoacid sequence depicted in FIG. 2 and as set forth in SEQ ID NO:12; andb) a VL variant 1 amino acid sequence depicted in FIG. 6 and as setforth in SEQ ID NO:20.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 2 aminoacid sequence depicted in FIG. 2 and as set forth in SEQ ID NO:12; andb) a VL variant 2 amino acid sequence depicted in FIG. 7 and as setforth in SEQ ID NO:22.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 2 aminoacid sequence depicted in FIG. 2 and as set forth in SEQ ID NO:12; andb) a VL variant 5 amino acid sequence depicted in FIG. 8 and as setforth in SEQ ID NO:24.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 3 aminoacid sequence depicted in FIG. 3 and as set forth in SEQ ID NO:14; andb) a VL variant 1 amino acid sequence depicted in FIG. 6 and as setforth in SEQ ID NO:20.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 3 aminoacid sequence depicted in FIG. 3 and as set forth in SEQ ID NO:14; andb) a VL variant 2 amino acid sequence depicted in FIG. 7 and set forthin SEQ ID NO:22. In some embodiments, the anti-C1s antibody comprises:a) a VH region comprising an amino acid sequence having at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% percent identity with SEQ ID NO: 14; and b) a VL regioncomprising an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% percentidentity with SEQ ID NO: 22. In some embodiments, the anti-C1s antibodycomprises: a) a VH region comprising the amino acid sequence of SEQ IDNO: 14; and b) a VL region comprising the amino acid sequence of SEQ IDNO: 22.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 3 aminoacid sequence depicted in FIG. 3 and as set forth in SEQ ID NO:14; andb) a VL variant 5 amino acid sequence depicted in FIG. 8 and as setforth in SEQ ID NO: 24.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 4 aminoacid sequence depicted in FIG. 4 and as set forth in SEQ ID NO:16; andb) a VL variant 1 amino acid sequence depicted in FIG. 6 and as setforth in SEQ ID NO:20.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 4 aminoacid sequence depicted in FIG. 4 and as set forth in SEQ ID NO:16; andb) a VL variant 2 amino acid sequence depicted in FIG. 7 and as setforth in SEQ ID NO:22.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 4 aminoacid sequence depicted in FIG. 4 and as set forth in SEQ ID NO:16; andb) a VL variant 5 amino acid sequence depicted in FIG. 8 and as setforth in SEQ ID NO: 24.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 5 aminoacid sequence depicted in FIG. 5 and as set forth in SEQ ID NO:18; andb) a VL variant 1 amino acid sequence depicted in FIG. 6 and as setforth in SEQ ID NO:20.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 5 aminoacid sequence depicted in FIG. 5 and as set forth in SEQ ID NO:18; andb) a VL variant 2 amino acid sequence depicted in FIG. 7 and as setforth in SEQ ID NO:22.

In some cases, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure comprises: a) a VH variant 5 aminoacid sequence depicted in FIG. 5 and as set forth in SEQ ID NO:18; andb) a VL variant 5 amino acid sequence depicted in FIG. 8 and as setforth in SEQ ID NO:24.

In particular embodiments, the antibody comprises a VH region comprisingSEQ ID NO:14, a VL region comprising SEQ ID NO:22, and a heavy chainconstant region; wherein the heavy chain constant region comprises anIgG4 constant region, wherein amino acid residue 308 of the heavy chainconstant region corresponding to SEQ ID NO: 28 is a leucine, and aminoacid residue 314 of the heavy chain constant region corresponding to SEQID NO: 28 is a serine; and wherein the antibody specifically bindsactivated C1s. In another embodiment, the antibody comprises a VH regioncomprising SEQ ID NO:14, a VL region comprising SEQ ID NO:22, and aheavy chain constant region comprising SEQ ID NO:28; wherein theantibody specifically binds activated C1s.

In some embodiments, the anti-C1s antibody comprises a heavy chaincomprising an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% identityto SEQ ID NO: 29. As one non-limiting example, in some cases, anantibody, e.g., a humanized anti-C1s antibody, of the present disclosurecomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO:29.

In some embodiments, the anti-C1s antibody comprises a light chaincomprising an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% identityto SEQ ID NO: 30. As one non-limiting example, in some cases, anantibody, e.g., a humanized anti-C1s antibody, of the present disclosurecomprises a light chain comprising the amino acid sequence set forth inSEQ ID NO:30.

In some embodiments, the antibody comprises a heavy chain comprising SEQID NO:29 and a light chain comprising SEQ ID NO:30.

In some embodiments, the anti-C1s antibody comprises: a) a heavy chaincomprising an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% identityto SEQ ID NO:29; and b) a light chain comprising an amino acid sequencehaving at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity to SEQ ID NO:30. In some cases, anantibody, e.g., a humanized anti-C1s antibody, of the present disclosurecomprises: a) a heavy chain comprising the amino acid sequence set forthin SEQ ID NO:29; and b) a light chain comprising the amino acid sequenceset forth in SEQ ID NO:30. In some embodiments, to produce such anantibody, a nucleic acid(s) comprising a nucleotide sequence(s) encodingthe amino acid sequences set forth in SEQ ID NO:31 and SEQ ID NO:32 canbe used.

In certain embodiments the anti-C1s antibody comprises a light chainconstant region. In some embodiments, the light chain constant regioncomprises SEQ ID NO: 45(RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

Binding Affinity

In some cases, an antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure binds a complement C1s protein from an individualthat has a complement system. In some embodiments, an antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure binds acomplement C1s protein from a mammal, fish, or invertebrate that has acomplement system. In some embodiments, an antibody, e.g., a humanizedanti-C1s antibody, of the present disclosure binds a mammaliancomplement C1s protein. In some embodiments, an antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure binds a humancomplement C1s protein. In some embodiments, an antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure binds a ratcomplement C1s protein. In some embodiments, an antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure binds acomplement C1s protein having the amino acid sequence depicted in FIG.13 (SEQ ID NO:9). Amino acid sequence SEQ ID NO:9 represents Homosapiens complement C1s protein, which has the amino acid sequence setforth in FIG. 13.

In some cases, an antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure binds a complement C1s protein with a dissociationconstant (K_(D)) of no more than 2.5 nM. In some embodiments, anantibody, e.g., a humanized anti-C1s antibody, of the present disclosurebinds a complement C1s protein with a K_(D) of no more than 2 nM. Insome embodiments, an antibody, e.g., a humanized anti-C1s antibody, ofthe present disclosure binds a complement C1s protein with a K_(D) of nomore than 1.5 nM. In some embodiments, an antibody, e.g., a humanizedanti-C1s antibody, of the present disclosure binds a complement C1sprotein with a K_(D) of no more than 1 nM. In some embodiments, anantibody, e.g., a humanized anti-C1s antibody, of the present disclosurebinds a complement C1s protein with a K_(D) of no more than 0.9 nM, nomore than 0.8 nM, no more than 0.7 nM, no more than 0.6 nM, no more than0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM,no more than 0.1 nM. In some cases, an antibody, e.g., a humanizedanti-C1s antibody, of the present disclosure binds a complement C1sprotein with a K_(D) of no more than 0.3 nM. In some cases, an antibody,e.g., a humanized anti-C1s antibody, of the present disclosure binds acomplement C1s protein with a K_(D) of no more than 0.2 nM. In somecases, an antibody, e.g., a humanized anti-C1s antibody of the presentdisclosure, binds a complement C1s protein with a K_(D) of no more than0.1 nM. Methods to measure binding of an antibody to C1s protein can bedetermined by one skilled in the art.

In some cases, an antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure binds a complement C1s protein with a K_(D) of nomore than 90 pM, no more than 80 pM, no more than 70 pM, no more than 60pM, no more than 50 pM, no more than 40 pM, no more than 30 pM, no morethan 20 pM, no more than 10 pM, no more than 9 pM, no more than 8 pM, nomore than 7 pM, no more than 6 pM, no more than 5 pM, no more than 4 pM,no more than 3 pM, no more than 2 pM, no more than 1 pM.

In some cases, an antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure binds a human complement C1s protein with adissociation constant (K_(D)) of no more than 2.5 nM. In some cases, anantibody, e.g., a humanized anti-C1s antibody, of the present disclosurebinds a human complement C1s protein with a dissociation constant(K_(D)) of no more than 1.5 nM. In some embodiments, an antibody, e.g.,a humanized anti-C1s antibody, of the present disclosure binds a humancomplement C1s protein with a K_(D) of no more than 2 nM. In some cases,an antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure binds a human complement C1s protein with a K_(D) of no morethan 1 nM. In some cases, an antibody, e.g., a humanized anti-C1santibody, of the present disclosure binds a human complement C1s proteinwith a K_(D) of no more than 0.9 nM, no more than 0.8 nM, no more than0.7 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM,no more than 0.3 nM, no more than 0.2 nM, no more than 0.1 nM. In someembodiments, an antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure binds a human complement C1s protein with a K_(D) ofno more than 0.3 nM. In some cases, an antibody, e.g., a humanizedanti-C1s antibody, of the present disclosure binds a human complementC1s protein with a K_(D) of no more than 0.2 nM. In some cases, anantibody, e.g., a humanized anti-C1s antibody, of the present disclosurebinds a human complement C1s protein with a K_(D) of no more than 0.1nM. Methods to measure binding of an antibody to human C1s protein canbe determined by one skilled in the art. In some cases, a binding assayas described in the Examples is used to determine the K_(D) between anantibody and a human C1s protein.

In some cases, an antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure binds a human complement C1s protein with a K_(D) ofno more than 90 pM, no more than 80 pM, no more than 70 pM, no more than60 pM, no more than 50 pM, no more than 40 pM, no more than 30 pM, nomore than 20 pM, no more than 10 pM, no more than 9 pM, no more than 8pM, no more than 7 pM, no more than 6 pM, no more than 5 pM, no morethan 4 pM, no more than 3 pM, no more than 2 pM, no more than 1 pM.

In some cases, an antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure inhibits the classical complement pathway with ahalf-maximal inhibitory concentration (IC₅₀) of 10⁻⁸ M or less, 5×10⁻⁹ Mor less, or 10⁻⁹ M or less.

An antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, when administered to an individual in need thereof, canreduce complement pathway (CP) activity from 10% to 100% (e.g., from 10%to 15%, from 15% to 20%, from 20% to 25%, from 25% to 30%, from 30% to40%, from 40% to 50%, from 50% to 60%, from 60% to 70%, from 70% to 80%,from 80% to 90%, or from 90% to 100%) for a period of time of from 1 dayto 1 week, from 1 week to 2 weeks, from 2 weeks to 4 weeks, from 4 weeksto 2 months, or more than 2 months.

For example, in some cases, a single dose of an antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure, whenadministered to an individual in need thereof, can reduce CP activityfrom 10% to 100% (e.g., from 10% to 15%, from 15% to 20%, from 20% to25%, from 25% to 30%, from 30% to 40%, from 40% to 50%, from 50% to 60%,from 60% to 70%, from 70% to 80%, from 80% to 90%, or from 90% to 100%)for a period of time of from 1 day to 1 week, from 1 week to 2 weeks,from 2 weeks to 4 weeks, from 4 weeks to 2 months, or more than 2months.

An antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, when administered to an individual in need thereof, canprovide for a serum concentration of the humanized anti-C1s antibodythat is effective to reduce CP activity from 10% to 100% (e.g., from 10%to 15%, from 15% to 20%, from 20% to 25%, from 25% to 30%, from 30% to40%, from 40% to 50%, from 50% to 60%, from 60% to 70%, from 70% to 80%,from 80% to 90%, or from 90% to 100%) for a period of time of from 1 dayto 1 week, from 1 week to 2 weeks, from 2 weeks to 4 weeks, from 4 weeksto 2 months, or more than 2 months.

Nucleic Acids, Expression Vectors, and Host Cells

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure. In some cases, a nucleic acid ofthe present disclosure comprises a nucleotide sequence encoding the VHregion of an anti-C1s antibody, e.g., a humanized anti-C1s antibody, ofthe present disclosure. In some cases, a nucleic acid of the presentdisclosure comprises a nucleotide sequence encoding the VL region of ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure. In some cases, a nucleic acid of the present disclosurecomprises a nucleotide sequence encoding the VH region and the VL regionof an anti-C1s antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure.

A nucleotide sequence encoding an anti-C1s antibody, e.g., a humanizedanti-C1s antibody, of the present disclosure can be operably linked toone or more regulatory elements, such as a promoter and enhancer, thatallow expression of the nucleotide sequence in the intended target cells(e.g., a cell that is genetically modified to synthesize the encodedantibody). Thus, in some cases, the present disclosure provides anucleic acid comprising a nucleotide sequence encoding an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, where the nucleotide sequence is operably linked to one ormore regulatory elements, e.g., a promoter and/or an enhancer.

Suitable promoter and enhancer elements are known in the art. Suitablepromoters for use in prokaryotic host cells include, but are not limitedto, a bacteriophage T7 RNA polymerase promoter; a T3 promoter; a T5promoter; a lambda P promoter; a trp promoter; a lac operon promoter; ahybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybridpromoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tacpromoter, and the like; a gpt promoter; an araBAD promoter; in vivoregulated promoters, such as an ssaG promoter or a related promoter(see, e.g., U.S. Patent Publication No. 20040131637), apagC promoter(Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93;Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter(Harborne et al. (1992) Mol. Micro. 6:2805-2813), and the like (see,e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al.(2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter(see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); astationary phase promoter, e.g., a dps promoter, an spy promoter, andthe like; a promoter derived from the pathogenicity island SPI-2 (see,e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al.(2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g.,Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a tet promoter(see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. andHeinemann, U. (eds), Topics in Molecular and Structural Biology,Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp.143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl. AcidsRes. 12:7035); and the like. Suitable strong promoters for use inprokaryotes such as Escherichia coli include, but are not limited toTrc, Tac, T5, T7, and P_(Lambda). Non-limiting examples of operators foruse in bacterial host cells include a lactose promoter operator (LacIrepressor protein changes conformation when contacted with lactose,thereby preventing the LacI repressor protein from binding theoperator), a tryptophan promoter operator (when complexed withtryptophan, TrpR repressor protein has a conformation that binds theoperator; in the absence of tryptophan, the TrpR repressor protein has aconformation that does not bind the operator), and a tac promoteroperator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci.U.S.A. 80:21-25).

In some embodiments, e.g., for expression in a yeast cell, a suitablepromoter is a constitutive promoter such as an ADH1 promoter, a PGK1promoter, an ENO promoter, a PYK1 promoter and the like; or aregulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2promoter, a PHO5 promoter, a CUP1 promoter, a GAL7 promoter, a MET25promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1promoter, and AOX1 (e.g., for use in Pichia).

For expression in a eukaryotic cell, suitable promoters include, but arenot limited to, light and/or heavy chain immunoglobulin gene promoterand enhancer elements; cytomegalovirus immediate early promoter; herpessimplex virus thymidine kinase promoter; early and late SV40 promoters;promoter present in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters. Constitutive mammalian promoters include, but are not limitedto, the promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actinpromoter, and other constitutive promoters. Additional exemplary viralpromoters which function constitutively in eukaryotic cells include, forexample, promoters from the cytomegalovirus (CMV), simian virus (e.g.,SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV),Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) ofMoloney leukemia virus, and other retroviruses, and the thymidine kinasepromoter of herpes simplex virus. Other constitutive promoters are knownto those of ordinary skill in the art. The promoters useful as geneexpression sequences of the disclosure also include inducible promoters.Inducible promoters are expressed in the presence of an inducing agent.For example, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art.

A nucleic acid comprising a nucleotide sequence encoding an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the present disclosurecan be present in any expression vector and/or a cloning vector known inthe art. As used herein, an expression vector refers to any nucleic acidconstruct which contains the necessary elements for the transcriptionand translation of an inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation,when introduced into an appropriate host cell. Expression vectors caninclude plasmids, phagemids, viruses, and derivatives thereof.

Certain aspects of the present disclosure provide a recombinant vectorcomprising a nucleic acid comprising a nucleotide sequence encoding ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, where the recombinant vector is a cloning vector. Certainaspects of the present disclosure provide a recombinant vectorcomprising a nucleic acid comprising a nucleotide sequence encoding ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, where the recombinant vector is an expression vector, e.g.,where the nucleotide sequence is operably linked to appropriateregulatory sequence(s) in the expression vector to ensure expression ofthe encoded antibody. Where a subject antibody comprises two separatepolypeptides, nucleic acids encoding the two polypeptides can be clonedin the same or separate vectors to form one or more recombinant vectors.A recombinant vector can include a selectable marker, an origin ofreplication, and other features that provide for replication and/ormaintenance of the recombinant vector (e.g., recombinant expressionvector).

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating asubject recombinant vector. The following vectors are provided by way ofexample. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS,pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala,Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene)pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host can be present. Suitable expression vectorsinclude, but are not limited to, viral vectors. Examples of viralvectors include, but are not limited to, viral vectors based on:vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., InvestOpthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., HGene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see,e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al.,Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594,1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989)63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte etal., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; aretroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus,and vectors derived from retroviruses such as Rous Sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, human immunodeficiency virus (see,e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol73:7812 7816, 1999), myeloproliferative sarcoma virus, and mammary tumorvirus); and the like.

In some embodiments, the vector is a viral vector. Viral vectorsinclude, but are not limited to, nucleic acid sequences from thefollowing viruses: retrovirus, such as Moloney murine leukemia virus,Harvey murine sarcoma virus, murine mammary tumor virus, and Roussarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses;polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus;vaccinia virus; polio virus; and RNA virus such as a retrovirus. One canreadily employ other vectors well-known in the art. Certain viralvectors are based on non-cytopathic eukaryotic viruses in whichnon-essential genes have been replaced with the gene of interest.Non-cytopathic viruses include retroviruses, the life cycle of whichinvolves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. Retroviruseshave been approved for human gene therapy trials. Most useful are thoseretroviruses that are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell line withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H.Freeman Co., New York (1990) and Murry, E. J., Methods in MolecularBiology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

In one embodiment, the virus is an adeno-associated virus, adouble-stranded DNA virus. The adeno-associated virus can be engineeredto be replication-deficient and is capable of infecting a wide range ofcell types and species. It further has advantages such as heat and lipidsolvent stability; high transduction frequencies in cells of diverselineages, including hematopoietic cells; and lack of superinfectioninhibition thus allowing multiple series of transductions. Reportedly,the adeno-associated virus can integrate into human cellular DNA in asite-specific manner, thereby minimizing the possibility of insertionalmutagenesis and variability of inserted gene expression characteristicof retroviral infection. In addition, wild-type adeno-associated virusinfections have been followed in tissue culture for greater than 100passages in the absence of selective pressure, implying that theadeno-associated virus genomic integration is a relatively stable event.The adeno-associated virus can also function in an extrachromosomalfashion.

In another embodiment, the viral vector is an adeno-associated virus(AAV) that has been manipulated to carry a polynucleotide encoding ananti-C1s antibody as disclosed herein. General methods for obtainingrecombinant AAVs (rAAVs) have been disclosed. See, for example, U.S.Pat. No. 8,734,809, 2013/0195801 as well as the references citedtherein. In some embodiments, a rAAV vector comprises one or more AAVinverted terminal repeats (ITRs) and a transgene of interest (e.g., anoptimized FIX polynucleotide sequence). In certain embodiments, themethods of making rAAV involve culturing a desired host cell whichcontains a nucleic acid sequence encoding an AAV capsid protein orfragment thereof; a functional rep gene; a rAAV vector composed of, AAVinverted terminal repeats (ITRs) and a transgene of interest; andsufficient helper functions to permit packaging of the recombinant AAVvector into the AAV capsid proteins. Materials and methods forperforming these and related procedures have been disclosed, forexample, in U.S. Pat. No. 8,734,809, 2013/0195801, PCT/US1997/015692,PCT/US2002/033692, PCT/US2002/033630, WO2007/148971, WO00/20561,WO03/042361, and WO2007/04670.

One or more of different AAV vector sequences derived from nearly anyserotype can be used in accord with the present disclosure. Choice of aparticular AAV vector sequence will be guided by known parameters suchas tropism of interest, required vector yields, etc. Generally, the AAVserotypes have genomic sequences of significant homology at the aminoacid and the nucleic acid levels, provide a related set of geneticfunctions, produce virions which are related, and replicate and assemblesimilarly. For the genomic sequence of the various AAV serotypes and anoverview of the genomic similarities see e.g. GenBank Accession numberU89790; GenBank Accession number J01901; GenBank Accession numberAF043303; GenBank Accession number AF085716; Chlorini et al. (1997, J.Vir. 71: 6823-33); Srivastava et al. (1983, J. Vir. 45:555-64); Chloriniet al. (1999, J. Vir. 73:1309-1319); Rutledge et al. (1998, J. Vir.72:309-319); and Wu et al. (2000, J. Vir. 74: 8635-47). AAV serotypes 1,2, 3, 4 and 5 are an illustrative source of AAV nucleotide sequences foruse in the context of the present disclosure. AAV6, AAV7, AAV8 or AAV9or newly developed AAV-like particles obtained by e.g. capsid shufflingtechniques and AAV capsid libraries, or from newly designed, developedor evolved ITR's are also suitable for certain disclosure applications.See Dalkara, D et al. (2013), Sci Transl. Med. 5(189): 189ra76;Kotterman, M A Nat. Rev. Genet. (2014) 15(7):455.

In other embodiments, the vector is derived from lentivirus. In certainembodiments, the vector is a vector of a recombinant lentivirus capableof infecting non-dividing cells.

The lentiviral genome and the proviral DNA typically have the threegenes found in retroviruses: gag, pol and env, which are flanked by twolong terminal repeat (LTR) sequences. The gag gene encodes the internalstructural (matrix, capsid and nucleocapsid) proteins; the pol geneencodes the RNA-directed DNA polymerase (reverse transcriptase), aprotease and an integrase; and the env gene encodes viral envelopeglycoproteins. The 5′ and 3′ LTR's serve to promote transcription andpolyadenylation of the virion RNA's. The LTR contains all othercis-acting sequences necessary for viral replication. Lentiviruses haveadditional genes including vif, vpr, tat, rev, vpu, nef and vpx (inHIV-1, HIV-2 and/or SIV).

Adjacent to the 5′ LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsidation of viral RNA into particles (the Psi site). If thesequences necessary for encapsidation (or packaging of retroviral RNAinto infectious virions) are missing from the viral genome, the cisdefect prevents encapsidation of genomic RNA.

However, the resulting mutant remains capable of directing the synthesisof all virion proteins. The disclosure provides a method of producing arecombinant lentivirus capable of infecting a non-dividing cellcomprising transfecting a suitable host cell with two or more vectorscarrying the packaging functions, namely gag, pol and env, as well asrev and tat. As will be disclosed herein below, vectors lacking afunctional tat gene are desirable for certain applications. Thus, forexample, a first vector can provide a nucleic acid encoding a viral gagand a viral pol and another vector can provide a nucleic acid encoding aviral env to produce a packaging cell. Introducing a vector providing aheterologous gene, herein identified as a transfer vector, into thatpackaging cell yields a producer cell which releases infectious viralparticles carrying the foreign gene of interest.

According to the above-indicated configuration of vectors and foreigngenes, the second vector can provide a nucleic acid encoding a viralenvelope (env) gene. The env gene can be derived from nearly anysuitable virus, including retroviruses. In some embodiments, the envprotein is an amphotropic envelope protein which allows transduction ofcells of human and other species.

Examples of retroviral-derived env genes include, but are not limitedto: Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murinesarcoma virus (HaMuSV or HSV), murine mammary tumor virus (MuMTV orMMTV), gibbon ape leukemia virus (GaLV or GALV), human immunodeficiencyvirus (HIV) and Rous sarcoma virus (RSV). Other env genes such asVesicular stomatitis virus (VSV) protein G (VSV G), that of hepatitisviruses and of influenza also can be used.

The vector providing the viral env nucleic acid sequence is associatedoperably with regulatory sequences described elsewhere herein.

In certain embodiments, the vector includes a lentiviral vector in whichthe HIV virulence genes env, vif, vpr, vpu and nef were deleted withoutcompromising the ability of the vector to transduce non-dividing cells.

In some embodiments, the vector includes a lentiviral vector whichcomprises a deletion of the U3 region of the 3′ LTR. The deletion of theU3 region can be the complete deletion or a partial deletion.

In some embodiments, the lentiviral vector of the disclosure comprisinga nucleotide sequence encoding an anti-C1s antibody described herein canbe transfected in a cell with (a) a first nucleotide sequence comprisinga gag, a pol, or gag and pol genes and (b) a second nucleotide sequencecomprising a heterologous env gene; wherein the lentiviral vector lacksa functional tat gene. In other embodiments, the cell is furthertransfected with a fourth nucleotide sequence comprising a rev gene. Incertain embodiments, the lentiviral vector lacks functional genesselected from vif, vpr, vpu, vpx and nef, or a combination thereof.

In certain embodiments, a lentiviral vector comprises one or morenucleotide sequences encoding a gag protein, a Rev-response element, acentral polypurine track (cPPT), or any combination thereof.

Examples of the lentiviral vectors are disclosed in WO9931251,WO9712622, WO9817815, WO9817816, and WO9818934, which are incorporatedherein by reference in their entireties.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well-known to those of skill inthe art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been found to be particularlyadvantageous for delivering genes to cells in vivo because of theirinability to replicate within and integrate into a host genome. Theseplasmids, however, having a promoter compatible with the host cell, canexpress a peptide from a gene operably encoded within the plasmid. Somecommonly used plasmids available from commercial suppliers includepBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMVplasmids, pSV40, and pBlueScript. Additional examples of specificplasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro,catalog number V87020; pcDNA4/myc-His, catalog number V86320; andpBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad,Calif.). Other plasmids are well-known to those of ordinary skill in theart. Additionally, plasmids can be custom designed using standardmolecular biology techniques to remove and/or add specific fragments ofDNA.

Host Cells

The present disclosure provides isolated genetically modified host cells(e.g., in vitro cells) that are genetically modified with a subjectnucleic acid. In some embodiments, a subject isolated geneticallymodified host cell can produce a subject antibody. Such a cell isreferred to as a “recombinant cell” or a “genetically modified hostcell.” A genetically modified host cell of the present disclosurecomprises a nucleic acid comprising a nucleotide sequence encoding ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure.

Suitable host cells include eukaryotic host cells, such as a mammaliancell, an insect host cell, a yeast cell; and prokaryotic cells, such asa bacterial cell. Introduction of a subject nucleic acid into the hostcell can be effected, for example by calcium phosphate precipitation,DEAE dextran mediated transfection, liposome-mediated transfection,electroporation, or other known method.

Suitable mammalian cells include primary cells and immortalized celllines. Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), CVI (monkey kidneyline), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonickidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. Insome cases, the cells are HEK cells. In certain embodiments, the cellsare HEK 293 cells. In some cases, the cells are CHO cells, e.g., CHO-K1cells (ATCC No. CCL-61), CHO-M cells, CHO-DG44 cells (ATCC No.PTA-3356), DUXB11 (Chinese Hamster Ovary line, DHFR minus), R1610(Chinese hamster fibroblast), BALBC/3T3 (mouse fibroblast), HAK (hamsterkidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma),BFA-1clBPT (bovine endothelial cells), RAJI (human lymphocyte), PER.C6®,NS0, CAP, BHK21, and the like. In some embodiments, the host cell is aCOS cell. In some embodiments, the host cell is a 293 cell. In someembodiments, the host cell is a CHO cell.

Suitable yeast cells include, but are not limited to, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonasreinhardtii, and the like. In some embodiments, the host cell is aSaccharomyces. In some embodiments, the host cell is a Pichia.

Suitable prokaryotic cells include, but are not limited to, any of avariety of laboratory strains of Escherichia coli, Bacillus (e.g., B.subtilis), Lactobacillus sp., and the like. See, e.g., Carrier et al.(1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemoreet al. (1995) Science 270:299-302. Typically, the laboratory strain isone that is non-pathogenic. In some embodiments, the host cell isEscherichia coli. In some embodiments, the host cell is Bacillussubtilis.

Introduction of the isolated nucleic acid molecules of the disclosureinto the host cell can be accomplished by various techniques well knownto those of skill in the art. These include, but are not limited to,transfection (including electrophoresis and electroporation), protoplastfusion, calcium phosphate precipitation, cell fusion with enveloped DNA,microinjection, and infection with intact virus. See, Ridgway, A. A. G.“Mammalian Expression Vectors” Chapter 24.2, pp. 470-472 Vectors,Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Mostpreferably, plasmid introduction into the host is via electroporation.The transformed cells are grown under conditions appropriate to theproduction of the light chains and heavy chains, and assayed for heavyand/or light chain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orflourescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

Host cells comprising the isolated nucleic acid molecules of thedisclosure are grown in an appropriate growth medium. As used herein,the term “appropriate growth medium” means a medium containing nutrientsrequired for the growth of cells. Nutrients required for cell growth caninclude a carbon source, a nitrogen source, essential amino acids,vitamins, minerals, and growth factors. Optionally, the media cancontain one or more selection factors. Optionally the media can containbovine calf serum or fetal calf serum (FCS). In one embodiment, themedia contains substantially no IgG. The growth medium will generallyselect for cells containing the DNA construct by, for example, drugselection or deficiency in an essential nutrient which is complementedby the selectable marker on the DNA construct or co-transfected with theDNA construct. Cultured mammalian cells are generally grown incommercially available serum-containing or serum-free media (e.g., MEM,DMEM, DMEM/F12). In one embodiment, the medium is CDoptiCHO (Invitrogen,Carlsbad, Calif.). In another embodiment, the medium is CD17(Invitrogen, Carlsbad, Calif.). Selection of a medium appropriate forthe particular cell line used is within the level of those ordinaryskilled in the art.

Pharmaceutical Compositions

The present disclosure provides compositions, including pharmaceuticalcompositions comprising an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure. In general, a pharmaceuticalcomposition, also referred to herein as a formulation, comprises aneffective amount of an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure. An “effective amount” means adosage sufficient to produce a desired result, e.g., reduction in anadverse symptom associated with a complement-mediated disease ordisorder, amelioration of a symptom of a complement-mediated disease ordisorder, slowing progression of a complement-mediated disease ordisorder, etc. Generally, the desired result is at least a reduction ina symptom of a complement-mediated disease or disorder, as compared to acontrol. In some embodiments, an anti-C1s antibody, e.g., a humanizedanti-C1s antibody, of the present disclosure is formulated and/ormodified to enable the antibody to cross the blood-brain barrier. Insome embodiments, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure is delivered in such a manner as toavoid the blood-brain barrier. In some embodiments, an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the present disclosureis formulated with an agent that facilitates crossing the blood-brainbarrier. In some embodiments, an anti-C1s antibody, e.g., a humanizedanti-C1s antibody, of the present disclosure is fused, directly orthrough a linker, to a compound that promotes the crossing of theblood-brain barrier.

Formulations

The pharmaceutical composition can be formulated for parenteraladministration (i.e. intravenous, subcutaneous, or intramuscular) bybolus injection. Formulations for injection can be presented in unitdosage form, e.g., in ampoules or in multidose containers with an addedpreservative. The compositions can take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., pyrogen free water.

In the subject methods, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure can be administered to the hostusing any convenient means capable of resulting in the desiredtherapeutic effect or diagnostic effect. Thus, the agent can beincorporated into a variety of formulations for therapeuticadministration. More particularly, an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure can be formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers, pharmaceutically acceptablediluents, or other pharmaceutically acceptable excipients and can beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants and aerosols. In someembodiments, a pharmaceutical composition comprises an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the present disclosureand a pharmaceutically acceptable excipient.

In pharmaceutical dosage forms, an anti-C1s antibody, e.g., a humanizedanti-C1s antibody, of the present disclosure can be administered in theform of their pharmaceutically acceptable salts, or they can also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

An anti-C1s antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure can be formulated into preparations for injection bydissolving, suspending or emulsifying the antibody in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, propyleneglycol, synthetic aliphatic acid glycerides, injectable organic esters(e.g., ethyl oleate), esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Furthermore, the pharmaceuticalcomposition of the present disclosure can comprise further agents suchas dopamine or psychopharmacologic drugs, depending on the intended useof the pharmaceutical composition.

Pharmaceutical compositions comprising an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure are prepared bymixing an anti-C1s antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure having the desired degree of purity with optionalphysiologically acceptable carriers, other excipients, stabilizers,surfactants, buffers and/or tonicity agents. Acceptable carriers, otherexcipients and/or stabilizers are nontoxic to recipients at the dosagesand concentrations employed, and include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid,glutathione, cysteine, methionine and citric acid; preservatives (suchas ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methylor propyl parabens, benzalkonium chloride, or combinations thereof);amino acids such as arginine, glycine, ornithine, lysine, histidine,glutamic acid, aspartic acid, isoleucine, leucine, alanine,phenylalanine, tyrosine, tryptophan, methionine, serine, proline andcombinations thereof; monosaccharides, disaccharides and othercarbohydrates; low molecular weight (less than about 10 residues)polypeptides; proteins, such as gelatin or serum albumin; chelatingagents such as EDTA; sugars such as trehalose, sucrose, lactose,glucose, mannose, maltose, galactose, fructose, sorbose, raffinose,glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid;and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, orpolyethylene glycol (PEG).

The pharmaceutical composition can be in a liquid form, a lyophilizedform, or a liquid form reconstituted from a lyophilized form, whereinthe lyophilized preparation is to be reconstituted with a sterilesolution prior to administration. The standard procedure forreconstituting a lyophilized composition is to add back a volume of purewater (typically equivalent to the volume removed duringlyophilization); however solutions comprising antibacterial agents canbe used for the production of pharmaceutical compositions for parenteraladministration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

Exemplary antibody concentrations in a subject pharmaceuticalcomposition can range from about 1 mg/mL to about 200 mg/mL or fromabout 50 mg/mL to about 200 mg/mL, or from about 150 mg/mL to about 200mg/mL.

An aqueous formulation of an anti-C1s antibody, e.g., a humanizedanti-C1s antibody, of the present disclosure can be prepared in apH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0,or from about 5.0 to about 6.0, or alternatively about 5.5. Examples ofbuffers that are suitable for a pH within this range include phosphate-,histidine-, citrate-, succinate-, acetate-buffers and other organic acidbuffers. The buffer concentration can be from about 1 mM to about 100mM, or from about 5 mM to about 50 mM, depending, e.g., on the bufferand the desired tonicity of the formulation.

A tonicity agent can be included in the antibody formulation to modulatethe tonicity of the formulation. Exemplary tonicity agents includesodium chloride, potassium chloride, glycerin and any component from thegroup of amino acids, sugars as well as combinations thereof. In someembodiments, the aqueous formulation is isotonic, although hypertonic orhypotonic solutions can be suitable. The term “isotonic” denotes asolution having the same tonicity as some other solution with which itis compared, such as a physiological salt solution or serum. Tonicityagents can be used in an amount of about 5 mM to about 350 mM, e.g., inan amount of 100 mM to 350 nM.

A surfactant can also be added to the antibody formulation to reduceaggregation of the formulated antibody and/or minimize the formation ofparticulates in the formulation and/or reduce adsorption. Exemplarysurfactants include polyoxyethylensorbitan fatty acid esters (Tween),polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers(Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer,Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitablepolyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (soldunder the trademark TWEEN 20™) and polysorbate 80 (sold under thetrademark Tween 80™). Examples of suitable polyethylene-polypropylenecopolymers are those sold under the names PLURONIC® F68 or POLOXAMER188™. Examples of suitable Polyoxyethylene alkyl ethers are those soldunder the trademark BRIJ™. Exemplary concentrations of surfactant canrange from about 0.001% to about 1% w/v.

A lyoprotectant can also be added in order to protect the labile activeingredient (e.g. a protein) against destabilizing conditions during thelyophilization process. For example, known lyoprotectants include sugars(including glucose and sucrose); polyols (including mannitol, sorbitoland glycerol); and amino acids (including alanine, glycine and glutamicacid). Lyoprotectants can be included in an amount of about 10 mM to 500nM.

In some cases, a subject formulation includes an anti-C1s antibody,e.g., a humanized anti-C1s antibody, of the present disclosure, and oneor more of the above-identified agents (e.g., a surfactant, a buffer, astabilizer, a tonicity agent) and is essentially free of one or morepreservatives, such as ethanol, benzyl alcohol, phenol, m-cresol,p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, andcombinations thereof. In other embodiments, a preservative is includedin the formulation, e.g., at concentrations ranging from about 0.001 toabout 2% (w/v).

For example, a subject formulation can be a liquid or lyophilizedformulation suitable for parenteral administration, and can comprise:about 1 mg/mL to about 200 mg/mL of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure; about 0.001% toabout 1% of at least one surfactant; about 1 mM to about 100 mM of abuffer; optionally about 10 mM to about 500 mM of a stabilizer; andabout 5 mM to about 305 mM of a tonicity agent; and has a pH of about4.0 to about 7.0.

As another example, a subject parenteral formulation is a liquid orlyophilized formulation comprising: about 1 mg/mL to about 200 mg/mL ofan anti-C1s antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure; 0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mMSucrose; and has a pH of 5.5.

As another example, a subject parenteral formulation comprises alyophilized formulation comprising: 1) 15 mg/mL of a subject antibody(e.g., a humanized anti-C1s antibody of the present disclosure); 0.04%Tween 20 w/v; 20 mM L-histidine; and 250 mM sucrose; and has a pH of5.5; or 2) 75 mg/mL of a subject antibody; 0.04% Tween 20 w/v; 20 mML-histidine; and 250 mM sucrose; and has a pH of 5.5; or 3) 75 mg/mL ofa subject antibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mMsucrose; and has a pH of 5.5; or 4) 75 mg/mL of a subject antibody;0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM trehalose; and has apH of 5.5; or 5) 75 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 20mM L-histidine; and 250 mM trehalose; and has a pH of 5.5.

As another example, a subject parenteral formulation is a liquidformulation comprising: 1) 7.5 mg/mL of a subject antibody; 0.02% Tween20 w/v; 120 mM L-histidine; and 250 125 mM sucrose; and has a pH of 5.5;or 2) 37.5 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 10 mML-histidine; and 125 mM sucrose; and has a pH of 5.5; or 3) 37.5 mg/mLof a subject antibody; 0.01% Tween 20 w/v; 10 mM L-histidine; and 125 mMsucrose; and has a pH of 5.5; or 4) 37.5 mg/mL of a subject antibody;0.02% Tween 20 w/v; 10 mM L-histidine; 125 mM trehalose; and has a pH of5.5; or 5) 37.5 mg/mL of a subject antibody; 0.01% Tween 20 w/v; 10 mML-histidine; and 125 mM trehalose; and has a pH of 5.5; or 6) 5 mg/mL ofa subject antibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mMtrehalose; and has a pH of 5.5; or 7) 75 mg/mL of a subject antibody;0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mM mannitol; and has a pHof 5.5; or 8) 75 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 20 mML histidine; and 140 mM sodium chloride; and has a pH of 5.5; or 9) 150mg/mL of a subject antibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and250 mM trehalose; and has a pH of 5.5; or 10) 150 mg/mL of a subjectantibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mM mannitol;and has a pH of 5.5; or 11) 150 mg/mL of a subject antibody; 0.02% Tween20 w/v; 20 mM L-histidine; and 140 mM sodium chloride; and has a pH of5.5; or 12) 10 mg/mL of a subject antibody; 0.01% Tween 20 w/v; 20 mML-histidine; and 40 mM sodium chloride; and has a pH of 5.5.

A subject antibody can be utilized in aerosol formulation to beadministered via inhalation. A subject antibody can be formulated intopressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like. Aerosol formulations such as nasal sprayformulations include purified aqueous or other solutions of the activeagent with preservative agents and isotonic agents. Such formulationsare adjusted to a pH and isotonic state compatible with the nasal mucousmembranes.

Unit dosage forms for oral administration such as syrups, elixirs, andsuspensions can be provided wherein each dosage unit, for example,teaspoonful, tablespoonful, or tablet, contains a predetermined amountof the composition. Similarly, unit dosage forms for injection orintravenous administration can comprise a subject antibody in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically acceptable diluent,carrier or vehicle. The specifications for a subject antibody can dependon the particular antibody employed and the effect to be achieved, andthe pharmacodynamics associated with each antibody in the host.

Other modes of administration will also find use with a method of thepresent disclosure. For instance, a subject antibody can be formulatedin suppositories and, in some cases, aerosol and intranasalcompositions. For suppositories, the vehicle composition will includetraditional binders and carriers such as, polyalkylene glycols, ortriglycerides. Such suppositories can be formed from mixtures containingthe active ingredient in the range of about 0.5% to about 10% (w/w),e.g., about 1% to about 2%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed. The nasal formulations can also containpreservatives such as, but not limited to, chlorobutanol andbenzalkonium chloride. A surfactant can be present to enhance absorptionof the subject antibody by the nasal mucosa.

A subject antibody can be administered as an injectable formulation.Typically, injectable compositions are prepared as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared. The preparationcan also be emulsified or the antibody encapsulated in liposomevehicles.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of a subject antibody adequate toachieve the desired state in the subject being treated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

In some embodiments, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure is formulated in a controlledrelease formulation. Sustained-release preparations can be preparedusing methods well known in the art. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody in which the matrices arein the form of shaped articles, e.g. films or microcapsules. Examples ofsustained-release matrices include polyesters, copolymers of L-glutamicacid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,hydrogels, polylactides, degradable lactic acid-glycolic acid copolymersand poly-D-(−)-3-hydroxybutyric acid. Possible loss of biologicalactivity and possible changes in immunogenicity of antibodies comprisedin sustained-release preparations can be prevented by using appropriateadditives, by controlling moisture content and by developing specificpolymer matrix compositions.

Controlled release within the scope of the present disclosure can betaken to mean any one of a number of extended release dosage forms. Thefollowing terms can be considered to be substantially equivalent tocontrolled release, for the purposes of the present disclosure:continuous release, controlled release, delayed release, depot, extendedrelease, gradual release, immediate release, long-term release,programmed release, prolonged release, proportionate release, protractedrelease, repository, retard, slow release, spaced release, sustainedrelease, time coat, timed release, delayed action, extended action,layered-time action, long acting, prolonged action, repeated action,slowing acting, sustained action, and sustained-action medications.Further discussions of these terms can be found in Lesczek Krowczynski,Extended-Release Dosage Forms, 1987 (CRC Press, Inc.).

The various controlled release technologies cover a very broad spectrumof drug dosage forms. Controlled release technologies include, but arenot limited to physical systems and chemical systems.

Physical systems include, but are not limited to, reservoir systems withrate-controlling membranes, such as microencapsulation,macroencapsulation, and membrane systems; reservoir systems withoutrate-controlling membranes, such as hollow fibers, ultra microporouscellulose triacetate, and porous polymeric substrates and foams;monolithic systems, including those systems physically dissolved innon-porous, polymeric, or elastomeric matrices (e.g., nonerodible,erodible, environmental agent ingression, and degradable), and materialsphysically dispersed in non-porous, polymeric, or elastomeric matrices(e.g., nonerodible, erodible, environmental agent ingression, anddegradable); laminated structures, including reservoir layers chemicallysimilar or dissimilar to outer control layers; and other physicalmethods, such as osmotic pumps, or adsorption onto ion-exchange resins.

Chemical systems include, but are not limited to, chemical erosion ofpolymer matrices (e.g., heterogeneous, or homogeneous erosion), orbiological erosion of a polymer matrix (e.g., heterogeneous, orhomogeneous). Additional discussion of categories of systems forcontrolled release can be found in Agis F. Kydonieus, Controlled ReleaseTechnologies: Methods, Theory and Applications, 1980 (CRC Press, Inc.).

There are a number of controlled release drug formulations that aredeveloped for oral administration. These include, but are not limitedto, osmotic pressure-controlled gastrointestinal delivery systems;hydrodynamic pressure-controlled gastrointestinal delivery systems;membrane permeation-controlled gastrointestinal delivery systems, whichinclude microporous membrane permeation-controlled gastrointestinaldelivery devices; gastric fluid-resistant intestine targetedcontrolled-release gastrointestinal delivery devices; geldiffusion-controlled gastrointestinal delivery systems; andion-exchange-controlled gastrointestinal delivery systems, which includecationic and anionic drugs. Additional information regarding controlledrelease drug delivery systems can be found in Yie W. Chien, Novel DrugDelivery Systems, 1992 (Marcel Dekker, Inc.).

Dosages

A suitable dosage can be determined by an attending physician or otherqualified medical personnel, based on various clinical factors. As iswell known in the medical arts, dosages for any one patient depend uponmany factors, including the patient's size, body surface area, age, theparticular compound to be administered, sex of the patient, time, androute of administration, general health, and other drugs beingadministered concurrently. An anti-C1s antibody, e.g., a humanizedanti-C1s antibody of the present disclosure can be administered inamounts between 1 ng/kg body weight and 20 mg/kg body weight per dose,e.g. between 0.1 mg/kg body weight to 10 mg/kg body weight, e.g.,between 0.5 mg/kg body weight to 5 mg/kg body weight; however, dosesbelow or above this exemplary range are envisioned, especiallyconsidering the aforementioned factors. If the regimen is a continuousinfusion, it can also be in the range of 1 μg to 10 mg per kilogram ofbody weight per minute.

In some embodiments, a dose of an anti-C1s antibody, e.g., a humanizedanti-C1s antibody, of the present disclosure is in the range of 0.001 μgto 1000 μg; however, doses below or above this exemplary range areenvisioned, especially considering the aforementioned factors. In someembodiments, the dosage can range, e.g., from about 0.0001 to 100 mg/kg,or from about 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg,0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.) body weight. For example dosages canbe 1 mg/kg body weight or 10 mg/kg body weight or within the range of1-10 mg/kg, or at least 1 mg/kg. Doses intermediate in the above rangesare also intended to be within the scope of the disclosure.

In some embodiments, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure is administered in an amount thatprovides for a peak serum concentration of from about 1 μg/ml to about 1mg/ml, e.g., from about 1 μg/ml to about 2.5 μg/ml, from about 2.5 μg/mlto about 5 μg/ml, from about 5 μg/ml to about 7.5 μg/ml, from about 7.5μg/ml to about 10 μg/ml, from about 10 μg/ml to about 25 μg/ml, fromabout 25 μg/ml to about 50 μg/ml, from about 50 μg/ml to about 100μg/ml, from about 100 μg/ml to about 250 μg/ml, from about 250 μg/ml toabout 500 μg/ml, from about 500 μg/ml to about 750 μg/ml, or from about750 μg/ml to about 1000 μg/ml. In some embodiments, a subject anti-C1santibody is administered in an amount that provides for a peak serumconcentration of greater than 1 mg/ml, e.g., from about 1 mg/ml to about2 mg/ml, from about 2 mg/ml to about 5 mg/ml, or from about 5 mg/ml toabout 10 mg/ml. A humanized antibody of the present disclosure can beadministered according to any schedule and for any period of time.

Those of skill will readily appreciate that dose levels andadministration schedules can vary as a function of the specificantibody, the severity of the symptoms and the susceptibility of thesubject to side effects. Preferred dosages and administration schedulesfor a given compound are readily determinable by those of skill in theart by a variety of means.

Routes of Administration

A subject antibody is administered to an individual using any availablemethod and route suitable for drug delivery, including in vivo and exvivo methods, as well as systemic and localized routes ofadministration.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, intrathecal,intracranial, subcutaneous, intradermal, topical, intravenous,intraperitoneal, intraarterial (e.g., via the carotid artery), spinal orbrain delivery, rectal, nasal, oral, and other enteral and parenteralroutes of administration. Routes of administration can be combined, ifdesired, or adjusted depending upon the antibody and/or the desiredeffect. A subject antibody composition can be administered in a singledose or in multiple doses. In some embodiments, a subject antibodycomposition is administered orally. In some embodiments, a subjectantibody composition is administered via an inhalational route. In someembodiments, a subject antibody composition is administeredintranasally. In some embodiments, a subject antibody composition isadministered locally. In some embodiments, a subject antibodycomposition is administered intracranially. In some embodiments, asubject antibody composition is administered intravenously. In someembodiments, a subject antibody composition is administeredsubcutaneously. In some embodiments, a subject antibody composition isadministered intramuscularly. In some embodiments, a subject antibodycomposition is administered intrathecally.

An antibody of the present disclosure can be administered to a hostusing any available conventional methods and routes suitable fordelivery of conventional drugs, including systemic or localized routes.In general, routes of administration contemplated by the disclosureinclude, but are not necessarily limited to, enteral, parenteral, orinhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, intrathecal, and intravenous routes, i.e., any route ofadministration other than through the alimentary canal. Parenteraladministration can be carried to effect systemic or local delivery of asubject antibody. Where systemic delivery is desired, administrationtypically involves invasive or systemically absorbed topical or mucosaladministration of pharmaceutical preparations.

A subject antibody can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

By treatment is meant at least an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g., symptom, associated with thepathological condition being treated, such as a complement-mediateddisease or disorder. As such, treatment also includes situations wherethe pathological condition, or at least symptoms associated therewith,are completely inhibited, e.g., prevented from happening, or stopped,e.g., terminated, such that the host no longer suffers from thepathological condition, or at least the symptoms that characterize thepathological condition.

In some embodiments, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure is administered by injection and/ordelivery, e.g., to a site in a brain artery or directly into braintissue. A subject antibody, e.g., a humanized antibody, can also beadministered directly to a target site e.g., by biolistic delivery tothe target site.

A variety of hosts (wherein the term “host” is used interchangeablyherein with the terms “subject,” “individual,” and “patient”) aretreatable according to the subject methods. Generally such hosts are“mammals” or “mammalian,” where these terms are used broadly to describeorganisms which are within the class mammalia, including the orderscarnivore (e.g., cats), herbivores (e.g., cattle, horses, and sheep),omnivores (e.g., dogs, goats, and pigs), rodentia (e.g., mice, guineapigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys).In some embodiments, the host is an individual that has a complementsystem, such as a mammal, fish, or invertebrate. In some embodiments,the host is a complement system-containing mammal, fish, or invertebratecompanion animal, agricultural animal, work animal, zoo animal, or labanimal. In some embodiments, the host is human.

The embodiments include compositions comprising a container suitable forcontaining a composition comprising a subject anti-C1s antibody foradministration to an individual. For example, a subject antibody can bedisposed within a container suitable for containing a pharmaceuticalcomposition. The container can be, for example, a bottle (e.g., with aclosure device, such as a cap), a blister pack (e.g., which can providefor enclosure of one or more doses per blister), a vial, flexiblepackaging (e.g., sealed Mylar or plastic bags), an ampule (for singledoses in solution), a dropper, a syringe, thin film, a tube and thelike. In some embodiments, a container, such as a sterile container,comprises a subject pharmaceutical composition. In some embodiments thecontainer is a bottle or a syringe. In some embodiments the container isa bottle. In some embodiments the container is a syringe.

Kits with unit doses of an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure, e.g. in oral or injectable doses,are provided. In such kits, in addition to the containers containing theunit doses will be an informational package insert describing the useand attendant benefits of the antibody in treating pathologicalcondition of interest. Preferred compounds and unit doses are thosedescribed herein above.

Methods of Treating a Complement-Mediated Disease or Disorder

The present disclosure provides methods of treating a subject in needthereof with an anti-C1s antibody or a nucleotide encoding an anti-C1santibody of the present disclosure. In some embodiments, the methodscomprise treating a complement-mediated disease or disorder. The methodsgenerally involve administering an effective amount of an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, or a pharmaceutical composition comprising such an antibody,to an individual in need thereof. In some cases, administration of asubject anti-C1s antibody modulates the activity of complement C1s in acell, a tissue, a fluid, or an organ of an individual, and treats thecomplement-mediated disease or disorder.

Certain aspects of the present disclosure provide methods of inhibitingactivation of complement component C4 in an individual, the methodscomprising administering to the individual an effective amount of ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure or a pharmaceutical composition comprising such an antibody.The present disclosure provides methods of inhibiting complement C1sactivity in an individual, the methods comprising administering to theindividual an effective amount of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure or apharmaceutical composition comprising such an antibody. The presentdisclosure provides methods of reducing the level of a complementcomponent cleavage product in an individual (e.g., in a fluid, tissue,or organ in an individual), the methods comprising administering to theindividual an effective amount of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure or apharmaceutical composition comprising such an antibody.

In some cases, a method of the present disclosure to treat an individualhaving a complement-mediated disease or disorder comprises administeringto the individual an effective amount of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure or an effectiveamount of a pharmaceutical composition comprising: a) an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure; and b) a pharmaceutically acceptable excipient suitable foradministration to such individual. In some embodiments, the individualis a mammal. In some embodiments, the individual is a human.Administration can be by any route known to those skilled in the art,including those disclosed herein. In some embodiments, administering isintravenous. In some embodiments, administering is intrathecal. In someembodiments, administering is subcutaneous. In some embodiments,administering intramuscular.

In some cases, an “effective amount” of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure, or an “effectiveamount” of a subject pharmaceutical composition comprising an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, is an amount that, when administered in one or more doses toan individual in need thereof, reduces the level of a complementcomponent cleavage product in the individual (e.g., in a fluid, tissue,or organ in the individual). In some cases, an “effective amount” of ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, or an “effective amount” of a subject pharmaceuticalcomposition comprising an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure, is an amount that, whenadministered in one or more doses to an individual in need thereof,reduces the level of a complement component cleavage product in theindividual (e.g., in a fluid, tissue, or organ in the individual) by atleast about 1%, by at least about 5%, by at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, or about 100%, compared to the level of thecomplement component cleavage product in the fluid, tissue, or organ inthe absence of treatment with the anti-C1s antibody, e.g., beforetreatment with the anti-C1s antibody. In some embodiments, theindividual is a mammal. In some embodiments, the individual is a human.Administering can be by any route known to those skilled in the art,including those disclosed herein. In some embodiments, administering isintravenous. In some embodiments, the route of administration isintrathecal. In some embodiments, the route of administration isintravenous. In some embodiments, the route of administration issubcutaneous. In some embodiments, the route of administration isintramuscular.

In some cases, an “effective amount” of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure, or an “effectiveamount” of a subject pharmaceutical composition comprising an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, is an amount that, when administered in one or more doses toan individual in need thereof, reduces the activity of the classicalcomplement pathway in the individual (e.g., in a fluid, tissue, or organin the individual). In some cases, an “effective amount” of an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, or an “effective amount” of a subject pharmaceuticalcomposition comprising an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure, is an amount that, whenadministered in one or more doses to an individual in need thereof,reduces, within about 48 hours, within about 24 hours, within about 12hours, within about 8 hours, or within about 4 hours of administrationof the anti-C1s antibody, the activity of the classical complementpathway in the individual (e.g., in a fluid, tissue, or organ in theindividual), by at least about 1%, at least about 5%, at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%, compared to theactivity of the classical complement pathway in the fluid, tissue, ororgan in the absence of treatment with the anti-C1s antibody, e.g.,before treatment with the anti-C1s antibody. In some embodiments, theindividual is a mammal. In some embodiments, the individual is a human.Administration of the anti-C1s antibody, e.g., the humanized anti-C1santibody, can be by any route known to those skilled in the art,including those disclosed herein. In some embodiments, the route ofadministration is intrathecal. In some embodiments, the route ofadministration is intravenous. In some embodiments, the route ofadministration is subcutaneous. In some embodiments, the route ofadministration is intramuscular. The level of activity of the classicalcomplement pathway can be determined using any of a variety of methods.As one non-limiting example, the activity of the classical complementpathway can be determined ex vivo, e.g., by determining the level ofactivity of the classical complement pathway in a blood, serum, orplasma sample obtained from the individual. For example, the classicalcomplement pathway in the blood, serum, or plasma sample can beactivated ex vivo, and the amount of a complement component cleavageproduct (such as C5b-9) generated by such activation can be determined.

In some cases, an “effective amount” of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure, or an “effectiveamount” of a subject pharmaceutical composition comprising an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, is an amount that, when administered in one or more doses toan individual in need thereof, reduces the activity of the classicalcomplement pathway in the individual (e.g., in a fluid, tissue, or organin the individual). In some cases, an “effective amount” of an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, or an “effective amount” of a subject pharmaceuticalcomposition comprising an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure, is an amount that, whenadministered in one or more doses to an individual in need thereof,reduces, within about 48 hours, within about 24 hours, within about 12hours, within about 8 hours, or within about 4 hours of administrationof the anti-C1s antibody, the level of activity of the classicalcomplement pathway in the individual (e.g., in a fluid, tissue, or organin the individual), by at least about 1%, at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or about 100%, compared to thelevel of the activity of the classical complement pathway in the fluid,tissue, or organ in the absence of treatment with the anti-C1s antibody,e.g., before treatment with the anti-C1s antibody. In some embodiments,the individual is a mammal. In some embodiments, the individual is ahuman. Administration of the anti-C1s antibody, e.g., the humanizedanti-C1s antibody, can be by any route known to those skilled in theart, including those disclosed herein. In some embodiments, the route ofadministration is intrathecal. In some embodiments, the route ofadministration is intravenous. In some embodiments, the route ofadministration is subcutaneous. In some embodiments, the route ofadministration is intramuscular.

In some cases, an “effective amount” of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure, or an “effectiveamount” of a subject pharmaceutical composition comprising an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, is an amount that, when administered in one or more doses toan individual in need thereof, reduces the activity of the classicalcomplement pathway in the individual (e.g., in a fluid, tissue, or organin the individual). In some cases, an “effective amount” of an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, or an “effective amount” of a subject pharmaceuticalcomposition comprising an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure, is an amount that, whenadministered in one or more doses to an individual in need thereof,maintains a reduction in the level of activity of the classicalcomplement pathway in the individual (e.g., in a fluid, tissue, or organin the individual) of at least about 1%, at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or about 100%, compared to thelevel of the activity of the classical complement pathway in the fluid,tissue, or organ in the absence of treatment with the anti-C1s antibody,e.g., before treatment with the anti-C1s antibody, where the reductionis maintained for a period of time of from about 4 hours to about 30days (e.g., from 4 hours to 8 hours, from 8 hours to 24 hours, from 2days to 4 days, from 4 days to 7 days, from 7 days to 14 days, from 14days to 21 days, or from 21 days to 30 days). In some embodiments, theindividual is a mammal. In some embodiments, the individual is a human.Administration of the anti-C1s antibody, e.g., the humanized anti-C1santibody, can be by any route known to those skilled in the art,including those disclosed herein. In some embodiments, the route ofadministration is intrathecal. In some embodiments, the route ofadministration is intravenous. In some embodiments, the route ofadministration is subcutaneous. In some embodiments, the route ofadministration is intramuscular.

In some cases, an “effective amount” of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure, or an “effectiveamount” of a subject pharmaceutical composition comprising an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, is an amount that, when administered in one or more doses toan individual in need thereof, reduces the activity of the classicalcomplement pathway in the individual (e.g., in a fluid, tissue, or organin the individual). In some cases, an “effective amount” of an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, or an “effective amount” of a subject pharmaceuticalcomposition comprising an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure, is an amount that, whenadministered in one or more doses to an individual in need thereof,maintains a reduction in the level of activity of the classicalcomplement pathway in the individual (e.g., in a fluid, tissue, or organin the individual) of at least about 1%, at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or about 100%, compared to thelevel of activity of the classical complement pathway in the fluid,tissue, or organ in the absence of treatment with the anti-C1s antibody,e.g., before treatment with the anti-C1s antibody, where the reductionis maintained for a period of time of from about 4 hours to about 21days (e.g., from 4 hours to 8 hours, from 8 hours to 24 hours, from 2days to 4 days, from 4 days to 7 days, from 7 days to 14 days, or from14 days to 21 days). In some embodiments, the individual is a mammal. Insome embodiments, the individual is a human. Administration of theanti-C1s antibody, e.g., the humanized anti-C1s antibody, can be by anyroute known to those skilled in the art, including those disclosedherein. In some embodiments, the route of administration is intrathecal.In some embodiments, the route of administration is intravenous. In someembodiments, the route of administration is subcutaneous. In someembodiments, the route of administration is intramuscular.

In some cases, an “effective amount” of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure, or an “effectiveamount” of a subject pharmaceutical composition comprising an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, is an amount that, when administered in one or more doses toan individual in need thereof, reduces the level of a complementcomponent cleavage product in the individual (e.g., in a fluid, tissue,or organ in the individual). In some cases, an “effective amount” of ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, or an “effective amount” of a subject pharmaceuticalcomposition comprising an anti-C1s antibody, e.g., a humanized anti-C1santibody of the present disclosure, is an amount that, when administeredin one or more doses to an individual in need thereof, reduces, withinabout 48 hours, within about 24 hours, within about 12 hours, withinabout 8 hours, or within about 4 hours of administration of the anti-C1santibody, e.g., the humanized anti-C1s antibody, the level of acomplement component cleavage product in the individual (e.g., in afluid, tissue, or organ in the individual), by at least about 1%, atleast about 5%, at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, orabout 100%, compared to the level of the complement component cleavageproduct in the fluid, tissue, or organ in the absence of treatment withthe anti-C1s antibody, e.g., before treatment with the anti-C1santibody. In some embodiments, the individual is a mammal. In someembodiments, the individual is a human. Administration of the anti-C1santibody, e.g., the humanized anti-C1s antibody can be by any routeknown to those skilled in the art, including those disclosed herein. Insome embodiments, the route of administration is intrathecal. In someembodiments, the route of administration is intravenous. In someembodiments, the route of administration is subcutaneous. In someembodiments, the route of administration is intramuscular.

In some cases, an “effective amount” of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure, or an “effectiveamount” of a subject pharmaceutical composition comprising an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, is an amount that, when administered in one or more doses toan individual in need thereof, reduces the level of a complementcomponent cleavage product in the individual (e.g., in a fluid, tissue,or organ in the individual). In some cases, an “effective amount” of ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, or an “effective amount” of a subject pharmaceuticalcomposition comprising an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure, is an amount that, whenadministered in one or more doses to an individual in need thereof,reduces, within about 48 hours, within about 24 hours, within about 12hours, within about 8 hours, or within about 4 hours of administrationof the anti-C1s antibody, the level of a complement component cleavageproduct in the individual (e.g., in a fluid, tissue, or organ in theindividual), by at least about 1%, at least about 5%, at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%, compared to thelevel of the complement component cleavage product in the fluid, tissue,or organ in the absence of treatment with the anti-C1s antibody, e.g.,before treatment with the anti-C1s antibody. In some embodiments, theindividual is a mammal. In some embodiments, the individual is a human.Administration of the anti-C1s antibody, e.g., the humanized anti-C1santibody, can be by any route known to those skilled in the art,including those disclosed herein. In some embodiments, the route ofadministration is intrathecal. In some embodiments, the route ofadministration is intravenous. In some embodiments, the route ofadministration is subcutaneous. In some embodiments, the route ofadministration is intramuscular.

In some cases, an “effective amount” of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure, or an “effectiveamount” of a subject pharmaceutical composition comprising an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, is an amount that, when administered in one or more doses toan individual in need thereof, reduces the level of a complementcomponent cleavage product in the individual (e.g., in a fluid, tissue,or organ in the individual). In some cases, an “effective amount” of ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, or an “effective amount” of a subject pharmaceuticalcomposition comprising an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure, is an amount that, whenadministered in one or more doses to an individual in need thereof,maintains a reduction in the level of a complement component cleavageproduct in the individual (e.g., in a fluid, tissue, or organ in theindividual) of at least about 1%, at least about 5%, at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, or about 100%, compared to the level ofthe complement component cleavage product in the fluid, tissue, or organin the absence of treatment with the anti-C1s antibody, e.g., beforetreatment with the anti-C1s antibody, where the reduction is maintainedfor a period of time of from about 4 hours to about 30 days (e.g., from4 hours to 8 hours, from 8 hours to 24 hours, from 2 days to 4 days,from 4 days to 7 days, from 7 days to 14 days, from 14 days to 21 days,or from 21 days to 30 days). In some embodiments, the individual is amammal. In some embodiments, the individual is a human. Administrationof the anti-C1s antibody, e.g., the humanized anti-C1s antibody, can beby any route known to those skilled in the art, including thosedisclosed herein. In some embodiments, the route of administration isintrathecal. In some embodiments, the route of administration isintravenous. In some embodiments, the route of administration issubcutaneous. In some embodiments, the route of administration isintramuscular.

In some cases, an “effective amount” of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure, or an “effectiveamount” of a subject pharmaceutical composition comprising an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, is an amount that, when administered in one or more doses toan individual in need thereof, reduces the level of a complementcomponent cleavage product in the individual (e.g., in a fluid, tissue,or organ in the individual). In some cases, an “effective amount” of ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, or an “effective amount” of a subject pharmaceuticalcomposition comprising an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure, is an amount that, whenadministered in one or more doses to an individual in need thereof,maintains a reduction in the level of a complement component cleavageproduct in the individual (e.g., in a fluid, tissue, or organ in theindividual) of at least about 1%, at least about 5%, at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, or about 100%, compared to the level ofthe complement component cleavage product in the fluid, tissue, or organin the absence of treatment with the anti-C1s antibody, e.g., beforetreatment with the anti-C1s antibody, where the reduction is maintainedfor a period of time of from about 4 hours to about 21 days (e.g., from4 hours to 8 hours, from 8 hours to 24 hours, from 2 days to 4 days,from 4 days to 7 days, from 7 days to 14 days, or from 14 days to 21days). In some embodiments, the individual is a mammal. In someembodiments, the individual is a human. Administration of the anti-C1santibody, e.g., the humanized anti-C1s antibody, can be by any routeknown to those skilled in the art, including those disclosed herein. Insome embodiments, the route of administration is intrathecal. In someembodiments, the route of administration is intravenous. In someembodiments, the route of administration is subcutaneous. In someembodiments, the route of administration is intramuscular.

In some cases, an “effective amount” of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure, or an “effectiveamount” of a subject pharmaceutical composition comprising an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, is an amount that, when administered in one or more doses toan individual in need thereof, reduces production of C4b2a (i.e.,complement C4b and C2a complex; also known as “C3 convertase”) in theindividual (or in a fluid, tissue, or organ of the individual) by atleast about 1%, at least about 5%, at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or about 100%, compared to the amount of C4b2a producedin the individual, or the fluid, tissue, or organ, in the absence oftreatment with the anti-C1s antibody, e.g., before treatment with theanti-C1s antibody. In some embodiments, the individual is a mammal. Insome embodiments, the individual is a human. Administering can be by anyroute known to those skilled in the art, including those disclosedherein. In some embodiments, administering is intravenous. In someembodiments, the route of administration is intrathecal. In someembodiments, the route of administration is intravenous. In someembodiments, the route of administration is subcutaneous. In someembodiments, the route of administration is intramuscular.

The present disclosure provides a method to modulate complementactivation. In some embodiments the method inhibits complementactivation, for example to reduce production of C4b2a. In someembodiments, the present disclosure provides a method to modulatecomplement activation in an individual having a complement-mediateddisease or disorder, the method comprising administering to theindividual an anti-C1s antibody, e.g., a humanized anti-C1s antibody, ofthe present disclosure or a pharmaceutical composition of the presentdisclosure, wherein the pharmaceutical composition comprises an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure. In some embodiments such a method inhibits complementactivation. In some embodiments, the individual is a mammal. In someembodiments, the individual is a human. Administering can be by anyroute known to those skilled in the art, including those disclosedherein. In some embodiments, administering is intravenous. In someembodiments, administering is intrathecal. In some embodiments,administering is subcutaneous. In some embodiments, the route ofadministration is intramuscular.

A complement-mediated disease or disorder is a disorder characterized byan abnormal amount of complement C1s or an abnormal level of complementC1s proteolytic activity in a cell, a tissue, a fluid, or an organ of anindividual.

In some cases, a complement-mediated disease or disorder ischaracterized by the presence in a cell, a tissue, or a fluid of anelevated (higher than normal) amount of C1s or of an elevated level ofcomplement C1s activity. For example, in some cases, acomplement-mediated disease or disorder is characterized by the presencein brain tissue and/or cerebrospinal fluid of an elevated amount and/oran elevated activity of C1s. A “higher than normal” amount of C1s in acell, a tissue, or a fluid indicates that the amount of C1s in the cell,tissue or fluid is higher than a normal, control level, e.g., higherthan a normal, control level for an individual or population ofindividuals of the same age group. A “higher than normal” level of C1sactivity in a cell, a tissue, an organ, or a fluid indicates that theproteolytic cleavage effected by C1s in the cell, tissue, organ, orfluid is higher than a normal, control level, e.g., higher than anormal, control level for an individual or population of individuals ofthe same age group. In some cases, an individual having acomplement-mediated disease or disorder exhibits one or more additionalsymptoms of such a disease or disorder.

In other cases, a complement-mediated disease or disorder ischaracterized by the presence in a cell, a tissue, or a fluid of a lowerthan normal amount of C1s or of a lower level of complement C1sactivity. For example, in some cases, a complement-mediated disease ordisorder is characterized by the presence in brain tissue and/orcerebrospinal fluid of a lower amount and/or a lower activity of C1s. A“lower than normal” amount of C1s in a cell, a tissue, or a fluidindicates that the amount of C1s in the cell, tissue or fluid is lowerthan a normal, control level, e.g., lower than a normal, control levelfor an individual or population of individuals of the same age group. A“lower than normal” level of C1s activity in a cell, a tissue, or afluid indicates that the proteolytic cleavage effected by C1s in thecell, tissue or fluid is lower than a normal, control level, e.g., lowerthan a normal, control level for an individual or population ofindividuals of the same age group. In some cases, an individual having acomplement-mediated disease or disorder exhibits one or more additionalsymptoms of such a disease or disorder.

A complement-mediated disease or disorder is a disease or disorder inwhich the amount or activity of complement C1s is such as to causedisease or disorder in an individual. In some embodiments, thecomplement-mediated disease or disorder is selected from the groupconsisting of alloimmune disease, autoimmune disease, cancer,hematological disease, infectious disease, inflammatory disease,ischemia-reperfusion injury, neurodegenerative disease,neurodegenerative disorder, ocular disease, renal disease, transplantrejection, vascular disease, and vasculitis disease. In someembodiments, the complement-mediated disease or disorder is anautoimmune disease. In some embodiments, the complement-mediated diseaseor disorder is an alloimmune disease. In some embodiments, thecomplement-mediated disease or disorder is cancer. In some embodiments,the complement-mediated disease or disorder is an infectious disease. Insome embodiments, the complement-mediated disease or disorder is aninflammatory disease. In some embodiments, the complement-mediateddisease or disorder is a hematological disease. In some embodiments, thecomplement-mediated disease or disorder is an ischemia-reperfusioninjury. In some embodiments, the complement-mediated disease or disorderis ocular disease. In some embodiments, the complement-mediated diseaseor disorder is a renal disease. In some embodiments, thecomplement-mediated disease or disorder is transplant rejection. In someembodiments, the complement-mediated disease or disorder isantibody-mediated transplant rejection. In some embodiments, thecomplement-mediated disease or disorder is a vascular disease. In someembodiments, the complement-mediated disease or disorder is a vasculitisdisorder. In some embodiments, the complement-mediated disease ordisorder is a neurodegenerative disease or disorder. In someembodiments, the complement-mediated disease is a neurodegenerativedisease. In some embodiments, the complement-mediated disorder is aneurodegenerative disorder.

Examples of a complement-mediated disease or disorder include, but arenot limited to, age-related macular degeneration, Alzheimer's disease,amyotrophic lateral sclerosis, anaphylaxis, argyrophilic grain dementia,arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis,atypical hemolytic uremic syndrome, autoimmune diseases (including,e.g., autoimmune hemolytic anemia (AIHA); warm AIHA; mixed AIHA; etc.),Barraquer-Simons syndrome, Behçet's disease, British type amyloidangiopathy, bullous pemphigoid, Buerger's disease, C1q nephropathy,cancer, catastrophic antiphospholipid syndrome, cerebral amyloidangiopathy, cold agglutinin disease, corticobasal degeneration,Creutzfeldt-Jakob disease, Crohn's disease, cryoglobulinemic vasculitis,dementia pugilistica, dementia with Lewy Bodies (DLB), diffuseneurofibrillary tangles with calcification, Discoid lupus erythematosus,Down's syndrome, Evan's syndrome, focal segmental glomerulosclerosis,formal thought disorder, frontotemporal dementia (FTD), frontotemporaldementia with parkinsonism linked to chromosome 17, frontotemporal lobardegeneration, Gerstmann-Straussler-Scheinker disease, Guillain-Barrésyndrome, Hallervorden-Spatz disease, hemolytic-uremic syndrome,hereditary angioedema, hypophosphastasis, idiopathic pneumonia syndrome,immune complex diseases, inclusion body myositis, infectious disease(e.g., disease caused by bacterial (e.g., Neisseria meningitidis orStreptococcus) viral (e.g., human immunodeficiency virus (HIV)), orother infectious agents), inflammatory disease, ischemia/reperfusioninjury, mild cognitive impairment, immunothrombocytopenic purpura (ITP),molybdenum cofactor deficiency (MoCD) type A, membranoproliferativeglomerulonephritis (MPGN) I, membranoproliferative glomerulonephritis(MPGN) II (dense deposit disease), membranous nephritis, multi-infarctdementia, lupus (e.g., systemic lupus erythematosus (SLE)),glomerulonephritis, Kawasaki disease, multifocal motor neuropathy,multiple sclerosis, multiple system atrophy, myasthenia gravis,myocardial infarction, myotonic dystrophy, neuromyelitis optica,Niemann-Pick disease type C, non-Guamanian motor neuron disease withneurofibrillary tangles, Parkinson's disease, Parkinson's disease withdementia, paroxysmal nocturnal hemoglobinuria, Pemphigus vulgaris,Pick's disease, postencephalitic parkinsonism, polymyositis, prionprotein cerebral amyloid angiopathy, progressive subcortical gliosis,progressive supranuclear palsy, psoriasis, sepsis, Shiga-toxin E coli(STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosingpanencephalitis, Tangle only dementia, transplant rejection, vasculitis(e.g., ANCA associated vasculitis), Wegner's granulomatosis, sickle celldisease, cryoglobulinemia, mixed cryoglobulinemia, essential mixedcryoglobulinemia, Type II mixed cryoglobulinemia, Type III mixedcryoglobulinemia, nephritis, drug-induced thrombocytopenia, lupusnephritis, Epidermolysis bullosa acquisita, delayed hemolytictransfusion reaction, hypocomplementemic urticarial vasculitis syndrome,pseudophakic bullous keratopathy, platelet refractoriness, chronicinflammatory demyelinating polyneuropathy (CIDP), myelodysplasticsyndrome (MDS), miller fisher syndrome, acute inflammatory demyelinatingpolyneuropathy (AIDP), acute motor axonal neuropathy (AMAN), acute motorand sensory axonal neuropathy (AMSAN), and pharyngeal-cervical-brachialvariant. In one embodiment, the complement-mediated disease or disordercomprises bullous pemphigoid. In one embodiment, the complement-mediateddisease or disorder comprises cold agglutinin disease. In oneembodiment, the complement-mediated disease or disorder comprisesautoimmune hemolytic anemia (AIHA). In one embodiment, thecomplement-mediated disease or disorder comprises immunothrombocytopenicpurpura (ITP). In one embodiment, the complement-mediated disease ordisorder comprises multifocal motor neuropathy. In one embodiment, thecomplement-mediated disease or disorder comprises neuromyelitis optica.

In some embodiments, the complement-mediated disease or disordercomprises Alzheimer's disease. In some embodiments, thecomplement-mediated disease or disorder comprises Parkinson's disease.In some embodiments, the complement-mediated disease or disordercomprises transplant rejection. In some embodiments, thecomplement-mediated disease or disorder is antibody-mediated transplantrejection.

In some embodiments, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure prevents or delays the onset of atleast one symptom of a complement-mediated disease or disorder in anindividual. In some embodiment, an anti-C1s antibody of the presentdisclosure reduces or eliminates at least one symptom of acomplement-mediated disease or disorder in an individual. Examples ofsymptoms include, but are not limited to, symptoms associated withautoimmune disease, cancer, hematological disease, infectious disease,inflammatory disease, ischemia-reperfusion injury, neurodegenerativedisease, neurodegenerative disorder, renal disease, transplantrejection, ocular disease, vascular disease, or a vasculitis disorder.The symptom can be a neurological symptom, for example, impairedcognitive function, memory impairment, loss of motor function, etc. Thesymptom can also be the activity of C1s protein in a cell, tissue, orfluid of an individual. The symptom can also be the extent of complementactivation in a cell, tissue, or fluid of an individual.

In some embodiments, administering an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure to an individualmodulates complement activation in a cell, tissue, or fluid of anindividual. In some embodiments, administration of a subject anti-C1santibody to an individual inhibits complement activation in a cell,tissue, or fluid of an individual. For example, in some embodiments, asubject anti-C1s antibody, e.g., a humanized anti-C1s antibody, whenadministered in one or more doses as monotherapy or in combinationtherapy to an individual having a complement-mediated disease ordisorder, inhibits complement activation in the individual by at leastabout 1%, at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, or 100%,compared to complement activation in the individual before treatmentwith the anti-C1s antibody.

In some embodiments, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure reduces C3 deposition onto red bloodcells; for example, in some embodiments, an anti-C1s antibody, e.g., ananti-C1s antibody, of the present disclosure reduces deposition of C3b,iC3b, etc., onto RBCs). In some embodiments, an anti-C1s antibody, e.g.,an anti-C1s antibody, of the present disclosure inhibitscomplement-mediated red blood cell lysis.

In some embodiments, an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure reduces C3 deposition ontoplatelets; for example, in some embodiments, an anti-C1s antibody, e.g.,an anti-C1s antibody, of the present disclosure reduces deposition ofC3b, iC3b, etc., onto platelets).

In some embodiments, administering an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure results in anoutcome selected from the group consisting of: (a) a reduction incomplement activation; (b) an improvement in cognitive function; (c) areduction in neuron loss; (d) a reduction in glial cell activation; (e)a reduction in lymphocyte infiltration; (f) a reduction in macrophageinfiltration; (g) a reduction in antibody deposition, (h) a reduction inglial cell loss; (i) a reduction in oligodendrocyte loss; (j) areduction in dendritic cell infiltration; (k) a reduction in neutrophilinfiltration; (1) a reduction in red blood cell lysis; (m) a reductionin red blood cell phagocytosis; (n) a reduction in plateletphagocytosis; (o) a reduction in platelet lysis; (p) an improvement intransplant graft survival; (q) a reduction in macrophage mediatedphagocytosis; (r) an improvement in vision; (s) an improvement in motorcontrol; (t) an improvement in thrombus formation; (u) an improvement inclotting; (v) an improvement in kidney function; (w) a reduction inantibody mediated complement activation; (x) a reduction in autoantibodymediated complement activation; (y) an improvement in anemia; (aa)reduction of demyelination; (ab) reduction of eosinophilia; (ac) areduction of C3 deposition on red blood cells (e.g., a reduction ofdeposition of C3b, iC3b, etc., onto RBCs); and (ad) a reduction in C3deposition on platelets (e.g., a reduction of deposition of C3b, iC3b,etc., onto platelets); and (ae) a reduction of anaphylatoxin toxinproduction; (af) a reduction in autoantibody mediated blister formation;(ag) a reduction in autoantibody induced pruritis; (ah) a reduction inautoantibody induced erythematosus; (ai) a reduction in autoantibodymediated skin erosion; (aj) a reduction in red blood cell destructiondue to transfusion reactions; (ak) a reduction in red blood cell lysisdue to alloantibodies; (al) a reduction in hemolysis due to transfusionreactions; (am) a reduction in allo-antibody mediated platelet lysis;(an) a reduction in platelet lysis due to transfusion reactions; (ao) areduction in mast cell activation; (ap) a reduction in mast cellhistamine release; (aq) a reduction in vascular permeability; (ar) areduction in edema; (as) a reduction in complement deposition ontransplant graft endothelium; (at) a reduction of anaphylatoxingeneration in transplant graft endothelium; (au) a reduction in theseparation of the dermal-epidermal junction; (av) a reduction in thegeneration of anaphylatoxins in the dermal-epidermal junction; (aw) areduction in alloantibody mediated complement activation in transplantgraft endothelium; (ax) a reduction in antibody mediated loss of theneuromuscular junction; (ay) a reduction in complement activation at theneuromuscular junction; (az) a reduction in anaphylatoxin generation atthe neuromuscular junction; (ba) a reduction in complement deposition atthe neuromuscular junction; (bb) a reduction in paralysis; (bc) areduction in numbness; (bd) increased bladder control; (be) increasedbowel control; (bf) a reduction in mortality associated withautoantibodies; (bg) a reduction in morbidity associated withautoantibodies; (bh) a reduction in Schwann cell damage; (bi) areduction in Schwann cell loss; (bj) a reduction in motor neuron damage;(bk) a reduction in motor neuron axonal loss; (bl) an amelioration ofaction potential conduction block; (bm) an improvement in upper or lowerlimb movement; (bn) an improvement in neuronal sensory-motor deficits,and (bo) any combination thereof.

In some embodiments, an anti-C1s antibody, e.g., a subject anti-C1santibody, when administered in one or more doses as monotherapy or incombination therapy to an individual having a complement-mediateddisease or disorder, is effect to achieve a reduction of at least about1%, at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or more than 90%, of one or more of thefollowing outcomes: (a) complement activation; (b) decline in cognitivefunction; (c) neuron loss; (d) glial cell activation; (e) lymphocyteinfiltration; (f) macrophage infiltration; (g) antibody deposition, (h)glial cell loss; (i) oligodendrocyte loss; (j) dendritic cellinfiltration; (k) neutrophil infiltration; (1) red blood cell lysis; (m)red blood cell phagocytosis; (n) platelet phagocytosis; (o) plateletlysis; (p) transplant graft rejection; (q) macrophage mediatedphagocytosis; (r) vision loss; (s) antibody mediated complementactivation; (t) autoantibody mediated complement activation; (u)demyelination; (v) eosinophilia; (w) or any combination thereof,compared to the level or degree of the outcome in the individual beforetreatment with the anti-C1s antibody.

In some embodiments, a subject anti-C1s antibody, when administered inone or more doses as monotherapy or in combination therapy to anindividual having a complement-mediated disease or disorder, is effectto achieve an improvement of at least about 1%, at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, or more than 90%, of one or more of the following outcomes: a)cognitive function; b) transplant graft survival; c) vision; d) motorcontrol; e) thrombus formation; f) clotting; g) kidney function; h)hematocrit (red blood cell count); and i) any combination thereof,compared to the level or degree of the outcome in the individual beforetreatment with the anti-C1s antibody.

In some embodiments, administering an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure to an individualreduces complement activation in the individual. For example, in someembodiments, a subject anti-C1s antibody, when administered in one ormore doses as monotherapy or in combination therapy to an individualhaving a complement-mediated disease or disorder, reduces complementactivation in the individual by at least about 1%, at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, or more than 90%, compared to complement activation in theindividual before treatment with the anti-C1s antibody.

In some embodiments, administering an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure improvescognitive function in the individual. For example, in some embodiments,a subject anti-C1s antibody, when administered in one or more doses asmonotherapy or in combination therapy to an individual having acomplement-mediated disease or disorder, improves cognitive function inthe individual by at least about 1%, at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, or morethan 90%, compared to the cognitive function in the individual beforetreatment with the anti-C1s antibody.

In some embodiments, administering an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure reduces the rateof decline in cognitive function in the individual. For example, in someembodiments, a subject anti-C1s antibody, when administered in one ormore doses as monotherapy or in combination therapy to an individualhaving a complement-mediated disease or disorder, reduces the rate ofdecline of cognitive function in the individual by at least about 1%, atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, or more than 90%, compared to the rate ofdecline in cognitive function in the individual before treatment withthe anti-C1s antibody.

In some embodiments, administering an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure to an individualreduces neuron loss in the individual. For example, in some embodiments,a subject anti-C1s antibody, when administered in one or more doses asmonotherapy or in combination therapy to an individual having acomplement-mediated disease or disorder, reduces neuron loss in theindividual by at least about 1%, at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, or more than90%, compared to neuron loss in the individual before treatment with theanti-C1s antibody.

In some embodiments, administering an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure to an individualreduces glial cell activation in the individual. For example, in someembodiments, a subject anti-C1s antibody, when administered in one ormore doses as monotherapy or in combination therapy to an individualhaving a complement-mediated disease or disorder, reduces glialactivation in the individual by at least about 1%, at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, or more than 90%, compared to glial cell activation in theindividual before treatment with the anti-C1s antibody. In someembodiments, the glial cells are astrocytes or microglia.

In some embodiments, administering an anti-C1s antibody, e.g., ahumanized anti-C1s antibody of the present disclosure to an individualreduces lymphocyte infiltration in the individual. For example, in someembodiments, a subject anti-C1s antibody, when administered in one ormore doses as monotherapy or in combination therapy to an individualhaving a complement-mediated disease or disorder, reduces lymphocyteinfiltration in the individual by at least about 1%, at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, or more than 90%, compared to lymphocyte infiltration in theindividual before treatment with the anti-C1s antibody.

In some embodiments, administering an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure to an individualreduces macrophage infiltration in the individual. For example, in someembodiments, an anti-C1s antibody, e.g., a humanized anti-C1s antibody,of the present disclosure, when administered in one or more doses asmonotherapy or in combination therapy to an individual having acomplement-mediated disease or disorder, reduces macrophage infiltrationin the individual by at least about 1%, at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,or more than 90%, compared to macrophage infiltration in the individualbefore treatment with the anti-C1s antibody.

In some embodiments, administering an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure to an individualreduces antibody deposition in the individual. For example, in someembodiments, an anti-C1s antibody, e.g., a humanized anti-C1s antibody,of the present disclosure, when administered in one or more doses asmonotherapy or in combination therapy to an individual having acomplement-mediated disease or disorder, reduces antibody deposition inthe individual by at least about 1%, at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, or morethan 90%, compared to antibody deposition in the individual beforetreatment with the anti-C1s antibody.

In some embodiments, administering an anti-C1s antibody of the presentdisclosure to an individual reduces anaphylatoxin (e.g., C3a, C4a, C5a)production in an individual. For example, in some embodiments, ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure, when administered in one or more doses as monotherapy or incombination therapy to an individual having a complement-mediateddisease or disorder, reduces anaphylatoxin production in the individualby at least about 1%, at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or more than 90%,compared to the level of anaphylatoxin production in the individualbefore treatment with the anti-C1s antibody.

The present disclosure provides for use of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure or apharmaceutical composition comprising an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure and apharmaceutically acceptable excipient to treat an individual having acomplement-mediated disease or disorder. In some embodiments, thepresent disclosure provides for use of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure to treat anindividual having a complement-mediated disease or disorder. In someembodiments, the present disclosure provides for use of a pharmaceuticalcomposition comprising an anti-C1s antibody, e.g., a humanized anti-C1santibody, of the present disclosure and a pharmaceutically acceptableexcipient to treat an individual having a complement-mediated disease ordisorder.

The present disclosure provides for use of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure in themanufacture of a medicament for the treatment of an individual having acomplement-mediated disease or disorder.

The present disclosure provides for use of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure or apharmaceutical composition comprising an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure and apharmaceutically acceptable excipient to inhibit complement activation.In some embodiments, the present disclosure provides for use of ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure or a pharmaceutical composition comprising an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the present disclosureand a pharmaceutically acceptable excipient to inhibit complementactivation in an individual having a complement-mediated disease ordisorder. In some embodiments, the present disclosure provides for useof an anti-C1s antibody, e.g., a humanized anti-C1s antibody, of thepresent disclosure to inhibit complement activation in an individualhaving a complement-mediated disease or disorder. In some embodiments,the present disclosure provides for use of a pharmaceutical compositioncomprising an anti-C1s antibody, e.g., a humanized anti-C1s antibody, ofthe present disclosure and a pharmaceutically acceptable excipient toinhibit complement activation in an individual having acomplement-mediated disease or disorder.

The present disclosure provides for use of an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure in themanufacture of a medicament for modulating complement activation. Insome embodiments, the medicament inhibits complement activation. In someembodiments, the medicament inhibits complement activation in anindividual having a complement-mediated disease or disorder.

The present disclosure provides for an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure or apharmaceutical composition comprising an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure and apharmaceutically acceptable excipient for use in medical therapy. Insome embodiments, the present disclosure provides for an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the present disclosurefor use in medical therapy. In some embodiments, the present disclosureprovides for a pharmaceutical composition comprising an anti-C1santibody, e.g., a humanized anti-C1s antibody, of the present disclosureand a pharmaceutically acceptable excipient for use in medical therapy.

The present disclosure provides for an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure or apharmaceutical composition comprising an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure and apharmaceutically acceptable excipient for treating an individual havinga complement-mediated disease or disorder. In some embodiments, thepresent disclosure provides for an anti-C1s antibody, e.g., a humanizedanti-C1s antibody, of the present disclosure for treating an individualhaving a complement-mediated disease or disorder. In some embodiments,the present disclosure provides for a pharmaceutical compositioncomprising an anti-C1s antibody, e.g., a humanized anti-C1s antibody, ofthe present disclosure and a pharmaceutically acceptable excipient fortreating an individual having a complement-mediated disease or disorder.

The present disclosure provides for an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure or apharmaceutical composition comprising an anti-C1s antibody, e.g., ahumanized anti-C1s antibody, of the present disclosure and apharmaceutically acceptable excipient for modulating complementactivation. In some embodiments, the present disclosure provides for ananti-C1s antibody, e.g., a humanized anti-C1s antibody, of the presentdisclosure for modulating complement activation. In some embodiments,the present disclosure provides for a pharmaceutical compositioncomprising an anti-C1s antibody, e.g., a humanized anti-C1s antibody, ofthe present disclosure and a pharmaceutically acceptable excipient formodulating complement activation. In some embodiments, the anti-C1santibody inhibits complement activation.

Examples of Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter describedabove can be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure numbered 1-37 areprovided below. As will be apparent to those of skill in the art uponreading this disclosure, each of the individually numbered aspects canbe used or combined with any of the preceding or following individuallynumbered aspects. This is intended to provide support for all suchcombinations of aspects and is not limited to combinations of aspectsexplicitly provided below:

Aspect 1. A humanized antibody that specifically binds complementcomponent C1s, wherein the antibody comprises:

-   -   a) a heavy chain comprising:    -   i) a VH region comprising the amino acid sequence:

(SEQ ID NO: 26) (Q/E)VQL(V/Q)QSGAE(V/L)KKPGASVK(L/V)SC(T/A)ASGFNIKDDYIHWV(K/R)QAPGQGLEWIGRIDPADGHTKYAPKFQVK(V/A)TITADTST(S/N)TAY(L/M)(E/Q)LSSL(R/T)SEDTAVYYCARYGYGREVFDYWGQG TTVTVSS.and

-   -   ii) an Fc region comprising an amino acid sequence having at        least 98% amino acid sequence identity with the amino acid        sequence set forth in SEQ ID NO:28, wherein amino acid 308 is        Leu and amino acid 314 is Ser; and    -   b) a light chain comprising:    -   i) a VL region comprising the amino acid sequence:

(SEQ ID NO: 27) DIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQK(T/P)GQPPK(I/L)LIYDASNLESGIPARFSGSGSGTDFTLTISSLE(E/P)EDFA(I/V)YYCQQSNEDPWTFGGGTKVEIK.and

-   -   ii) a light chain constant region.

Aspect 2. The humanized antibody of aspect 1, comprising: a) a VH regioncomprising SEQ ID NO:10; and b) a VL region comprising SEQ ID NO:20.

Aspect 3. The humanized antibody of aspect 1, comprising: a) a VH regioncomprising SEQ ID NO:10; and b) a VL region comprising SEQ ID NO:22.

Aspect 4. The humanized antibody of aspect 1, comprising: a) a VH regioncomprising SEQ ID NO:10; and b) a VL region comprising SEQ ID NO:24.

Aspect 5. The humanized antibody of aspect 1, comprising: a) a VH regioncomprising SEQ ID NO:12; and b) a VL region comprising SEQ ID NO:20.

Aspect 6 The humanized antibody of aspect 1, comprising: a) a VH regioncomprising SEQ ID NO:12; and b) a VL region comprising SEQ ID NO:22.

Aspect 7. The humanized antibody of aspect 1, comprising: a) a VH regioncomprising SEQ ID NO:12; and b) a VL region comprising SEQ ID NO:24.

Aspect 8. The humanized antibody of aspect 1, comprising: a) a VH regioncomprising SEQ ID NO:14; and b) a VL region comprising SEQ ID NO:20.

Aspect 9. The humanized antibody of aspect 1, comprising: a) a VH regioncomprising SEQ ID NO:14; and b) a VL region comprising SEQ ID NO:22.

Aspect 10. The humanized antibody of aspect 1, comprising: a) a VHregion comprising SEQ ID NO:14; and b) a VL region comprising SEQ IDNO:24.

Aspect 11. The humanized antibody of aspect 1, comprising: a) a VHregion comprising SEQ ID NO:16; and b) a VL region comprising SEQ IDNO:20.

Aspect 12. The humanized antibody of aspect 1, comprising: a) a VHregion comprising SEQ ID NO:16; and b) a VL region comprising SEQ IDNO:22.

Aspect 13. The humanized antibody of aspect 1, comprising: a) a VHregion comprising SEQ ID NO:16; and b) a VL region comprising SEQ IDNO:24.

Aspect 14. The humanized antibody of aspect 1, comprising: a) a VHregion comprising SEQ ID NO:18; and b) a VL region comprising SEQ IDNO:20.

Aspect 15. The humanized antibody of aspect 1, comprising: a) a VHregion comprising SEQ ID NO:18; and b) a VL region comprising SEQ IDNO:22.

Aspect 16. The humanized antibody of aspect 1, comprising: a) a VHregion comprising SEQ ID NO:18; and b) a VL region comprising SEQ IDNO:24.

Aspect 17. The humanized antibody of aspect 1, wherein the light chainconstant region is a human kappa light chain constant region.

Aspect 18. The humanized antibody of aspect 1, wherein the heavy chainconstant region comprises the amino acid sequence set forth in SEQ IDNO:28.

Aspect 19. The humanized antibody of aspect 1, wherein: a) the heavychain comprises the amino acid sequence set forth in SEQ ID NO:29; andb) the light chain comprises the amino acid sequence set forth in SEQ IDNO:30.

Aspect 20. A composition comprising: a) the humanized antibody of anyone of aspects 1-19; and b) a pharmaceutically acceptable excipient.

Aspect 21. A container comprising the composition of aspect 20.

Aspect 22. The container of aspect 21, wherein the container is sterile.

Aspect 23. The container of aspect 21 or aspect 22, wherein thecontainer is a vial, a bottle, or a syringe.

Aspect 24. A method of reducing the level of a complement componentcleavage product in an individual, the method comprising administeringto the individual the antibody of any one of aspects 1-19, or thecomposition of aspect 20, in an amount effective to inhibit C1s and toreduce the level of the cleavage product.

Aspect 25. The method of aspect 24, wherein the complement componentcleavage product is a C4 cleavage product.

Aspect 26. The method of aspect 25, wherein the complement componentcleavage product is a C2 cleavage product.

Aspect 27. The method of aspect 25, wherein the complement componentcleavage product is a C3 cleavage product.

Aspect 28. The method of any one of aspects 24-27, wherein theindividual is a human.

Aspect 29. The method of any one of aspects 24-28, wherein theadministering is intravenous.

Aspect 30. The method of any one of aspects 24-28, wherein theadministering is intramuscular.

Aspect 31. The method of any one of aspects 24-28, wherein theadministering is intrathecal.

Aspect 32. The method of any one of aspects 24-28, wherein theadministering is subcutaneous.

Aspect 33. The method of any one of aspects 24-28, wherein said reducingis effective to treat a complement-mediated disorder.

Aspect 34. The method of aspect 33, wherein the complement-mediateddisorder is an alloimmune disorder.

Aspect 35. The method of aspect 33, wherein the complement-mediateddisorder is an autoimmune disorder.

Aspect 36. A method of inhibiting C1s-mediated cleavage of a complementcomponent in an individual, the method comprising administering to theindividual the antibody of any one of aspects 1-19, or the compositionof aspect 20, in an amount effective to inhibit C1s-mediated cleavage ofa complement component.

Aspect 37. A method of treating a complement-mediated disease ordisorder in an individual, the method comprising administering to theindividual the antibody of any one of aspects 1-19, or the compositionof aspect 20, in an amount effective to treat the complement-mediateddisease or disorder.

Aspect 38. An antibody, comprising a heavy chain and a light chain,wherein the heavy chain comprises a VH region and a heavy chain constantregion, and the light chain comprises a VL region;

wherein the VL region comprises a VL CDR1, a VL CDR2, and a VL CDR3, and

-   -   wherein the VH region comprises a VH CDR1, a VH CDR2, and a VH        CDR3;    -   wherein the VL CDR1 comprises SEQ ID NO: 1;    -   wherein the VL CDR2 comprises SEQ ID NO: 2;    -   wherein the VL CDR3 comprises SEQ ID NO: 3;    -   wherein the VH CDR1 comprises SEQ ID NO: 4;    -   wherein the VH CDR2 comprises SEQ ID NO: 5;    -   wherein the VH CDR3 comprises SEQ ID NO: 6;    -   wherein the heavy chain constant region comprises an IgG4        constant region, wherein amino acid residue 308 of the heavy        chain constant region corresponding to SEQ ID NO: 28 is Leu, and        amino acid residue 314 of the heavy chain constant region        corresponding to SEQ ID NO: 28 is Ser;    -   and wherein the antibody specifically binds activated C1s.

Aspect 39. The antibody of aspect 38, wherein amino acid residue 108 ofthe heavy chain constant region corresponding to SEQ ID NO: 28 is Pro.

Aspect 40. The antibody of aspect 38 or 39, wherein amino acid residue115 of the heavy chain constant region corresponding to SEQ ID NO: 28 isGlu.

Aspect 41. An antibody, comprising a heavy chain and a light chain,wherein the heavy chain comprises a VH region and a heavy chain constantregion, and the light chain comprises a VL region;

wherein the VL region comprises a VL CDR1, a VL CDR2, and a VL CDR3, andwherein the VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3;

-   -   wherein the VL CDR1 comprises SEQ ID NO: 1;    -   wherein the VL CDR2 comprises SEQ ID NO: 2;    -   wherein the VL CDR3 comprises SEQ ID NO: 3;    -   wherein the VH CDR1 comprises SEQ ID NO: 4;    -   wherein the VH CDR2 comprises SEQ ID NO: 5;    -   wherein the VH CDR3 comprises SEQ ID NO: 6;    -   wherein the heavy chain constant region comprises SEQ ID NO: 28;    -   and wherein the antibody specifically binds activated C1s.

Aspect 42. The antibody of any one of aspects 38 to 41, wherein the VLregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 20, 22, and 24.

Aspect 43. The antibody of any one of aspects 38 to 42, wherein the VHregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 12, 14, 16, and 18.

Aspect 44. The antibody of any one of aspects 38 to 43, wherein:

(a) the VH region comprises SEQ ID NO:10, and the VL region comprisesSEQ ID NO:20;(b) the VH region comprises SEQ ID NO:10, and the VL region comprisesSEQ ID NO:22;(c) the VH region comprises SEQ ID NO:10, and the VL region comprisesSEQ ID NO:24;(d) the VH region comprises SEQ ID NO:12, and the VL region comprisesSEQ ID NO:20;(e) the VH region comprises SEQ ID NO:12, and the VL region comprisesSEQ ID NO:22;(f) the VH region comprises SEQ ID NO:12, and the VL region comprisesSEQ ID NO:24;(g) the VH region comprises SEQ ID NO:14, and the VL region comprisesSEQ ID NO:20;(h) the VH region comprises SEQ ID NO:14, and the VL region comprisesSEQ ID NO:22;(i) the VH region comprises SEQ ID NO:14, and the VL region comprisesSEQ ID NO:24;(j) the VH region comprises SEQ ID NO:16, and the VL region comprisesSEQ ID NO:20;(j) the VH region comprises SEQ ID NO:16, and the VL region comprisesSEQ ID NO:22;(k) the VH region comprises SEQ ID NO:16, and the VL region comprisesSEQ ID NO:24;(l) the VH region comprises SEQ ID NO:18, and the VL region comprisesSEQ ID NO:20;(m) the VH region comprises SEQ ID NO:18, and the VL region comprisesSEQ ID NO:22; or(n) the VH region comprises SEQ ID NO:18, and the VL region comprisesSEQ ID NO:24.

Aspect 45. The antibody of any one of aspects 38 to 44, wherein the VHregion comprises SEQ ID NO:14, and the VL region comprises SEQ ID NO:22.

Aspect 46. The antibody of any one of aspects 38 to 45, wherein thelight chain further comprises a light chain constant region.

Aspect 47. The antibody of aspect 46, wherein the light chain constantregion comprises SEQ ID NO: 45.

Aspect 48. The antibody of any one of aspects 38 to 47, where the heavychain comprises SEQ ID NO: 29.

Aspect 49. The antibody of any one of aspects 38 to 48, wherein thelight chain comprises SEQ ID NO: 30.

Aspect 50. The antibody of any one of aspects 1 to 19 and 38 to 49,which is a bispecific antibody or a multispecific antibody.

Aspect 51. An immunoconjugate comprising the antibody of any one ofaspects 1 to 19 and 38 to 50.

Aspect 52. A nucleotide of a set of nucleotides encoding the antibody ofany one of aspects 1 to 19 and 38 to 50.

Aspect 53. A vector or a set of vectors comprising the nucleotide of theset of nucleotides of aspect 52.

Aspect 54. A host cell comprising the nucleotide of the set ofnucleotides of aspect 52 or the vector or the set of vectors of aspect53.

Aspect 55. A pharmaceutical composition comprising the antibody of anyone of aspects 1 to 19 and 38 to 50, the immunoconjugate of aspect 51,the nucleotide or the set of nucleotides of aspect 52, the vector or theset of vectors of aspect 53, or the host cell of aspect 54, and apharmaceutically acceptable excipient.

Aspect 56. A method of inhibiting a complement pathway in a subject inneed thereof, comprising administering to the subject a pharmaceuticallyeffective amount of the antibody of any one of aspects 1 to 19 and 38 to50, the immunoconjugate of aspect 51, the nucleotide or the set ofnucleotides of aspect 52, the vector or the set of vectors of aspect 53,the host cell of aspect 54, or the pharmaceutical composition of aspect55.

Aspect 57. A method of inhibiting C1s-mediated cleavage of complementcomponent C4 in a subject in need thereof, comprising administering tothe subject a pharmaceutically effective amount of the antibody of anyone of aspects 1 to 19 and 38 to 50, the immunoconjugate of aspect 51,the nucleotide or the set of nucleotides of aspect 52, the vector or theset of vectors of aspect 53, the host cell of aspect 54, or thepharmaceutical composition of aspect 55.

Aspect 58. A method of treating a complement-mediated disease ordisorder in a subject in need thereof, comprising administering to thesubject a pharmaceutically effective amount of the antibody of any oneof aspects 1 to 19 and 38 to 50, the immunoconjugate of aspect 51, thenucleotide or the set of nucleotides of aspect 52, the vector or the setof vectors of aspect 53, the host cell of aspect 54, or thepharmaceutical composition of aspect 55.

Aspect 59. The method of aspect 58, wherein the complement-mediateddisease or disorder is selected from the group consisting of age-relatedmacular degeneration, Alzheimer's disease, amyotrophic lateralsclerosis, anaphylaxis, argyrophilic grain dementia, arthritis (e.g.,rheumatoid arthritis), asthma, atherosclerosis, atypical hemolyticuremic syndrome, autoimmune diseases (including, e.g., autoimmunehemolytic anemia (AIHA); warm AIHA; mixed AIHA; etc.), Barraquer-Simonssyndrome, Behçet's disease, British type amyloid angiopathy, bullouspemphigoid, Buerger's disease, C1q nephropathy, cancer, catastrophicantiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinindisease, corticobasal degeneration, Creutzfeldt-Jakob disease, Crohn'sdisease, cryoglobulinemic vasculitis, dementia pugilistica, dementiawith Lewy Bodies (DLB), diffuse neurofibrillary tangles withcalcification, Discoid lupus erythematosus, Down's syndrome, Evan'ssyndrome, focal segmental glomerulosclerosis, formal thought disorder,frontotemporal dementia (FTD), frontotemporal dementia with parkinsonismlinked to chromosome 17, frontotemporal lobar degeneration,Gerstmann-Straussler-Scheinker disease, Guillain-Barré syndrome,Hallervorden-Spatz disease, hemolytic-uremic syndrome, hereditaryangioedema, hypophosphastasis, idiopathic pneumonia syndrome, immunecomplex diseases, inclusion body myositis, infectious disease (e.g.,disease caused by bacterial (e.g., Neisseria meningitidis orStreptococcus) viral (e.g., human immunodeficiency virus (HIV)), orother infectious agents), inflammatory disease, ischemia/reperfusioninjury, mild cognitive impairment, immunothrombocytopenic purpura (ITP),molybdenum cofactor deficiency (MoCD) type A, membranoproliferativeglomerulonephritis (MPGN) I, membranoproliferative glomerulonephritis(MPGN) II (dense deposit disease), membranous nephritis, multi-infarctdementia, lupus (e.g., systemic lupus erythematosus (SLE)),glomerulonephritis, Kawasaki disease, multifocal motor neuropathy,multiple sclerosis, multiple system atrophy, myasthenia gravis,myocardial infarction, myotonic dystrophy, neuromyelitis optica,Niemann-Pick disease type C, non-Guamanian motor neuron disease withneurofibrillary tangles, Parkinson's disease, Parkinson's disease withdementia, paroxysmal nocturnal hemoglobinuria, Pemphigus vulgaris,Pick's disease, postencephalitic parkinsonism, polymyositis, prionprotein cerebral amyloid angiopathy, progressive subcortical gliosis,progressive supranuclear palsy, psoriasis, sepsis, Shiga-toxin E coli(STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosingpanencephalitis, Tangle only dementia, transplant rejection, vasculitis(e.g., ANCA associated vasculitis), Wegner's granulomatosis, sickle celldisease, cryoglobulinemia, mixed cryoglobulinemia, essential mixedcryoglobulinemia, Type II mixed cryoglobulinemia, Type III mixedcryoglobulinemia, nephritis, drug-induced thrombocytopenia, lupusnephritis, Epidermolysis bullosa acquisita, delayed hemolytictransfusion reaction, hypocomplementemic urticarial vasculitis syndrome,pseudophakic bullous keratopathy, platelet refractoriness, chronicinflammatory demyelinating polyneuropathy (CIDP), myelodysplasticsyndrome (MDS), miller fisher syndrome, acute inflammatory demyelinatingpolyneuropathy (AIDP), acute motor axonal neuropathy (AMAN), acute motorand sensory axonal neuropathy (AMSAN), pharyngeal-cervical-brachialvariant, and any combination thereof.

Aspect 60. The method of aspect 58 or 59, wherein thecomplement-mediated disease or disorder is selected from the groupconsisting of bullous pemphigoid, cold agglutinin disease, autoimmunehemolytic anemia (AIHA), immunothrombocytopenic purpura (ITP),multifocal motor neuropathy, neuromyelitis optica, and any combinationthereof.

Aspect 61. The method of any one of aspects 56 to 60, wherein theantibody is administered parenterally, intravenously, subcutaneously,intradermally, transdermally, intramuscularly, orally, intraocularly,intrathecally, intraperitoneally, intranasally, buccally, sublingually,rectally, vaginally, or via pulmonary route.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present disclosure, and are not intended to limit thescope of what the inventors regard as their disclosure nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations can be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Humanized Anti-aC1s Variants

Humanized variants of anti-aC1s were generated. Amino acid sequences ofthe heavy chain VH domains of humanized variants 1-5; nucleotidesequences encoding the heavy chain VH domain of the humanized variantsare also provided. Amino acid sequences of the light chain VL domain ofhumanized variants 1, 2, and 5, and nucleotide sequences encoding thelight chain VL domain of the humanized variants, are shown in FIGS. 6-8.Amino acid differences relative to the amino acid sequence of murineanti-aC1s (VL SEQ ID NO: 7; VH SEQ ID NO: 8) are summarized in FIGS. 9and 10.

Single letter amino acid codes are as follows (with 3-letter amino acidcodes in parentheses):

G—Glycine (Gly) P—Proline (Pro) A—Alanine (Ala) V—Valine (Val) L—Leucine(Leu) I—Isoleucine (Ile) M—Methionine (Met) C—Cysteine (Cys)F—Phenylalanine (Phe) Y—Tyrosine (Tyr) W—Tryptophan (Trp) H—Histidine(His) K—Lysine (Lys) R—Arginine (Arg) Q—Glutamine (Gln) N—Asparagine(Asn) E—Glutamic Acid (Glu) D—Aspartic Acid (Asp) S—Serine (Ser)T—Threonine (Thr) Example 2: Characterization of Humanized Anti-aC1sVariants

Binding characteristics of humanized anti-aC1s variants are provided inTables 4 and 5 (FIG. 11 and FIG. 12, respectively). The relative bindingaffinities for various humanized anti-aC1s variants to activated C1s areprovided in Table 4 (first data column), which is presented in FIG. 11.

All 15 combinations (VH variant 1+Vk variant 1; VH variant 1+Vk variant2; VH variant 1+Vk variant 5; VH variant 2+Vk variant 1; VH variant 2+Vkvariant 2; VH variant 2+Vk variant 5; VH variant 3+Vk variant 1; VHvariant 3+Vk variant 2; VH variant 3+Vk variant 5; VH variant 4+Vkvariant 1; VH variant 4+Vk variant 2; VH variant 4+Vk variant 5; VHvariant 5+Vk variant 1; VH variant 5+Vk variant 2; VH variant 5+Vkvariant 5) were produced. Each humanized variant was tested for theability to compete with biotinylated murine anti-aC1s for binding toactive C1s. The data are shown in FIG. 11, second data column.

Each humanized variant was tested in a commercially available assay thatmeasures complement classical pathway (CP) activation. The results areshown in FIG. 11, third data column. The data show that all 15 humanizedvariants inhibit CP activation with an IC₅₀ similar to that of murineanti-aC1s.

Kinetic characterization of binding affinity was carried out on 8 of thehumanized anti-aC1s variants. The data are depicted in Table 5, which ispresented in FIG. 12.

Example 3: In Vivo Studies in Cynomolgus Monkeys

To assess the pharmacokinetic (PK) and pharmacodynamic (PD) propertiesof humanized anti-aC1s, single-dose studies of humanized anti-aC1s wereperformed in cynomolgus monkeys (Macaca fascicularis). Additionally, tocompare the bioavailability of humanized anti-aC1s by various routes ofadministration, the humanized anti-aC1s variant was administered eitherby intravenous (IV) or subcutaneous (SC) injection. Following humanizedanti-aC1s dosing, plasma and serum samples were taken at designated timepoints to determine circulating concentrations of humanized anti-aC1s;and to assess inhibition of the classical complement pathway (CP) byhumanized anti-aC1s. Plasma and serum levels of humanized anti-aC1s overtime provide PK data; inhibition of the CP over time provides PD data.

All study animals were female, between 2.4-3.9 kg body weight, and werebetween the ages of 3-5 years old. Additionally, all animals were naïveto pharmaceutical dosing.

Whole blood was collected in K₂EDTA tubes and serum separator tubes forplasma and serum processing, respectively, and immediately stored at−15° C. to −25° C.

To assess the pharmacokinetic profile of the humanized anti-aC1s variantVH3/VK2-Fc-sub₄, plasma samples taken at the time points depicted inFIG. 13 and FIG. 14 were diluted and run in an ELISA to quantifyVH3/VK2-Fc-sub₄ plasma concentrations. Briefly, diluted plasma sampleswere added to a 96-well plate pre-coated with activated C1s. Followingplasma sample incubation and subsequent washing, a horseradishperoxidase-conjugated detection antibody specific for human IgG wasadded to detect C1s-bound VH3/VK2-Fc-sub₄. Finally, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added to initiate acolorimetric reaction that was read on a spectrophotometer. Byinterpolating from a standard curve of VH3/VK2-Fc-sub₄ run in parallelwith the plasma samples, VH3/VK2-Fc-sub₄ plasma concentrations weredetermined for all samples.

The pharmacodynamic effects of VH3/VK2-Fc-sub₄ were assessed using theWIESLAB® classical complement pathway kit. The WIESLAB® kit iscommercially available, and involves use of an enzyme-linkedimmunosorbent assay (ELISA) that is designed to evaluate the strength ofclassical complement pathway activity in serum samples by activating theclassical pathway of the sample ex vivo and measuring the ex vivogeneration of the final split product of the pathway, C5b-9. Sampleswere assayed according to the manufacturer's instructions. Briefly,serum samples from the monkeys, collected at the time points indicatedin FIG. 14 and FIG. 15, were diluted and added to the wells of theprovided 96-well plate. Following incubation, a detection antibodyspecific for the final split product of the classical pathway, C5b-9,was added and the colorimetric reaction measured on a spectrophotometer.All samples for an individual monkey were compared and normalized to thepre-dose sample of the same monkey (pre-dose=100% activity).

FIG. 13 depicts PD and PK data from 3 animals dosed withVH3/VK2-Fc-sub₄, administered intravenously at 10 mg/kg. As shown inFIG. 13, the serum concentration of VH3/VK2-Fc-sub₄ was between 70 μg/mLand 300 μg/mL over a period of time of up to 650 hours (27 days). Duringthis same time period, CP activity was inhibited 80% to 99%.

FIG. 14 depicts PD and PK data from 3 animals dosed withVH3/VK2-Fc-sub₄, administered subcutaneously at 20 mg/kg. As shown inFIG. 14, the serum concentration of VH3/VK2-Fc-sub₄ was between about 50μg/mL and about 450 μg/mL over a period of time of up to 85 days. Duringthe same time period, CP activity, as measured using the WEISLAB® kitwas inhibited 60% to 99%. Further, a single 20 mg/kg subcutaneous doseof VH3/VK2-Fc-sub₄ inhibited the complement pathway by greater than 90%for 28 days (FIG. 19).

Example 4: Characterization of an Anti-C1s Antibody Comprising aModified Fc Region

The humanized anti-aC1s variant, VH3/VK2, comprised a human IgG4, withS241P and L248E substitutions. To enhance half-life and subcutaneousavailability, the Fc region of VH3/VK2 was modified to include M428L andN434S substitutions. The resulting antibody, referred to asVH3/VK2-Fc-sub₄, was found to specifically bind active C1s with adissociation constant of 1.53×10⁹.

VH3/VK2-Fc-sub₄ inhibited the complement pathway in vitro to a similarextent as a general anti-C1s antibody, previously described as VH4/VK2in U.S. Pat. No. 8,945,562. In an in vitro classical complement pathwayserum activity assay, VH3/VK2-Fc-sub₄ (circles) displayed a similar ED₅₀as anti-C1s (squares), though the inhibition of classical complementpathway activity by VH3/VK2-Fc-sub₄ was more gradual than the inhibitionof complement activity by anti-C1s (FIG. 17A). In addition,VH3/VK2-Fc-sub₄ displayed similar levels of hemolysis as the generalanti-C1s antibody (FIG. 17B).

It was hypothesized that if FcRn binding is involved in the recycling ofVH3/VK2 (VH3/VK2-Fc-sub₄ without enhanced FcRn binding), thenVH3/VK2-Fc-sub₄ should have a longer half-life and therefore a prolongedpharmacodynamic effect compared to VH3/VK2. Cynomolgus monkeys wereadministered a single intravenous dose of 10 mg/kg of VH3/VK2 orVH3/VK2-Fc-sub₄, and blood was drawn periodically. Through 650 hourspost-administration, animals administered VH3/VK2-Fc-sub₄ hadconsistently higher levels of the antibody in their blood than animalsadministered VH3/VK2 (FIG. 18A). In addition, VH3/VK2-Fc-sub₄ had aprolonged pharmacodynamic effect than VH3/VK2. A single dose of 10 mg/kgof VH3/VK2-Fc-sub₄ inhibited complement pathway activity by more than70% through 650 hours, whereas the same dose of VH3/VK2 showed a gradualdecrease in inhibition, beginning almost immediately, and nearlyreaching the level of the vehicle control by 650 hours postadministration (FIG. 18B). These effects were also observed incynomolgus monkeys administered a single subcutaneous dose of 20 mg/kgVH3/VK2-Fc-sub₄ (see FIG. 14).

Example 5: Pharmacokinetic Study of Anti-C1s Antibody FollowingIntravenous and Subcutaneous Administrations in Cynomolgus Monkeys

The objective of this investigational study is to evaluate thepharmacokinetics of VH3/VK2-Fc-sub₄, following a single intravenous (IV)bolus injection, single IV bolus injection followed by once weeklysubcutaneous (SC) injections, or repeat SC injections in femalecynomolgus monkeys.

Study Design

Animals will be assigned to groups and treated as indicated in Table 2.Animals will either be dosed via intravenous (IV) bolus injection in aperipheral vein using primed butterfly infusion lines or by subcutaneous(SC) bolus injection in the interscapular region of the back. Ifirritation is noted at the injection site, the lower thoracic region canbe used for subsequent SC injections to avoid further irritation. Thefrequency of dosage is consistent with the anticipated pharmacokineticsof the test article. The regimen for treatment selected for this studyis anticipated to identify achievable concentrations in peripheral bloodand associated pharmacological activity.

TABLE 2 Group assignments Dosing Dose Number of Test Days/ LevelConcentration Volume^(a) Animals Group Material Frequency Route (mg/kg)(mg/mL) (mL/kg) Females 1 CA/V 1 IV 0 0 4 3 8, 15, 22, SC 0 0 2 29, 36,and 43 2 VH3/VK2- 1 IV 45 15 3 3 Fc-sub₄ 3 VH3/VK2- 1 IV 10 2.5 4 3Fc-sub₄ 8, 15, 22, SC 2 1 2 29, 36, and 43 4 VH3/VK2- 1 and 29 SC 10 5 23 Fc-sub₄ ^(a)Total dose volume (mL) will be calculated based on themost recent body weight. CA/V: control article/vehicle; IV: intravenousbolus injection; SC: subcutaneous bolus injection.

Clinical Observations

Clinical observations will be performed once daily, beginning on thesecond day of acclimation for each animal in the AM, prior to roomcleaning. A mortality check will be conducted twice daily to assessgeneral animal health and wellness.

Additional clinical observations can be performed, as necessary. Ifclinical observations for an animal demonstrate declining animalcondition, a veterinary evaluation will be performed and the StudyDirector notified.

Blood will be collected from a peripheral vein of restrained, consciousanimals. For the first 24 hours post dose, blood will not be collectedfrom the vein (or limb) that was used for IV dose administration. Bloodwill be collected via a single draw and then divided appropriately.

In the event of an unscheduled necropsy, venous blood samples will becollected from conscious moribund animals prior to anesthesia, ifpossible.

Blood samples will be collected at the following timepoints and storedon wet ice prior to processing:

Groups 1 and 3: Days 1 (15 min, 30 min, 1 hr, 2 hrs, and 4 hrs posdose),2 (24 hrs postdose), 5, 8 (predose, 30 min, 1 hr, 2 hrs, and 4 hrspostdose), 9, 10, 11, 12, 13, 14, 15 (predose), 18, 22 (predose), 25, 29(predose), 32, 36 (predose), 39, 43 (predose), 46, 50, 53, and 57;Group 2: Days 1 (15 min, 30 min, 1 hr, 2 hrs, and 4 hrs postdose), 2 (24hrs postdose), 3 (48 hrs postdose), 5 (96 hrs postdose), 8 (168 hrspostdose), 15, 22, 25, 29, 32, 36, 39, 43, 46, 50, 53, and 57; andGroup 4: Days 1 (30 min, 1 hr, 2 hrs, and 4 hrs postdose), 2 (24 hrspostdose), 3, 4, 5, 6, 11, 15, 18, 22, 25, 29 (predose, 30 min, 1 hr, 2hrs, and 4 hrs postdose), 30, 31, 32, 33, 34, 39, 43, 46, 50, 53, and57.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes can be made and equivalents can besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications can be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the claims appended hereto.

What is claimed is:
 1. An antibody, comprising a heavy chain and a lightchain, wherein the heavy chain comprises a heavy chain variable (VH)region and a heavy chain constant region, and the light chain comprisesa light chain variable (VL) region; wherein the VL region comprises a VLcomplementary determining region (CDR) 1, a VL CDR2, and a VL CDR3, andwherein the VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3;wherein the VL CDR1 comprises SEQ ID NO: 1; wherein the VL CDR2comprises SEQ ID NO: 2; wherein the VL CDR3 comprises SEQ ID NO: 3;wherein the VH CDR1 comprises SEQ ID NO: 4; wherein the VH CDR2comprises SEQ ID NO: 5; wherein the VH CDR3 comprises SEQ ID NO: 6;wherein the heavy chain constant region comprises an IgG4 constantregion, wherein amino acid residue 308 of the heavy chain constantregion corresponding to SEQ ID NO: 28 is Leu, and amino acid residue 314of the heavy chain constant region corresponding to SEQ ID NO: 28 isSer; and wherein the antibody specifically binds activated C1s.
 2. Theantibody of claim 1, wherein amino acid residue 108 of the heavy chainconstant region corresponding to SEQ ID NO: 28 is Pro.
 3. The antibodyof claim 1 or 2, wherein amino acid residue 115 of the heavy chainconstant region corresponding to SEQ ID NO: 28 is Glu.
 4. An antibody,comprising a heavy chain and a light chain, wherein the heavy chaincomprises a heavy chain variable (VH) region and a heavy chain constantregion, and the light chain comprises a light chain variable (VL)region; wherein the VL region comprises a VL complementary determiningregion (CDR) 1, a VL CDR2, and a VL CDR3, and wherein the VH regioncomprises a VH CDR1, a VH CDR2, and a VH CDR3; wherein the VL CDR1comprises SEQ ID NO: 1; wherein the VL CDR2 comprises SEQ ID NO: 2;wherein the VL CDR3 comprises SEQ ID NO: 3; wherein the VH CDR1comprises SEQ ID NO: 4; wherein the VH CDR2 comprises SEQ ID NO: 5;wherein the VH CDR3 comprises SEQ ID NO: 6; wherein the heavy chainconstant region comprises SEQ ID NO: 28; and wherein the antibodyspecifically binds activated C1s.
 5. The antibody of any one of claims 1to 4, wherein the VL region comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 20, 22, and
 24. 6. The antibodyof any one of claims 1 to 5, wherein the VH region comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 10, 12,14, 16, and
 18. 7. The antibody of any one of claims 1 to 6, wherein:(a) the VH region comprises SEQ ID NO:10, and the VL region comprisesSEQ ID NO:20; (b) the VH region comprises SEQ ID NO:10, and the VLregion comprises SEQ ID NO:22; (c) the VH region comprises SEQ ID NO:10,and the VL region comprises SEQ ID NO:24; (d) the VH region comprisesSEQ ID NO:12, and the VL region comprises SEQ ID NO:20; (e) the VHregion comprises SEQ ID NO:12, and the VL region comprises SEQ ID NO:22;(f) the VH region comprises SEQ ID NO:12, and the VL region comprisesSEQ ID NO:24; (g) the VH region comprises SEQ ID NO:14, and the VLregion comprises SEQ ID NO:20; (h) the VH region comprises SEQ ID NO:14,and the VL region comprises SEQ ID NO:22; (i) the VH region comprisesSEQ ID NO:14, and the VL region comprises SEQ ID NO:24; (j) the VHregion comprises SEQ ID NO:16, and the VL region comprises SEQ ID NO:20;(j) the VH region comprises SEQ ID NO:16, and the VL region comprisesSEQ ID NO:22; (k) the VH region comprises SEQ ID NO:16, and the VLregion comprises SEQ ID NO:24; (l) the VH region comprises SEQ ID NO:18,and the VL region comprises SEQ ID NO:20; (m) the VH region comprisesSEQ ID NO:18, and the VL region comprises SEQ ID NO:22; or (n) the VHregion comprises SEQ ID NO:18, and the VL region comprises SEQ ID NO:24.8. The antibody of any one of claims 1 to 7, wherein the VH regioncomprises SEQ ID NO:14, and the VL region comprises SEQ ID NO:22.
 9. Theantibody of any one of claims 1 to 8, wherein the light chain furthercomprises a light chain constant region.
 10. The antibody of claim 9,wherein the light chain constant region comprises SEQ ID NO:
 45. 11. Theantibody of any one of claims 1 to 10, where the heavy chain comprisesSEQ ID NO:
 29. 12. The antibody of any one of claims 1 to 11, whereinthe light chain comprises SEQ ID NO:
 30. 13. The antibody of any one ofclaims 1 to 12, which is a bispecific antibody or a multispecificantibody.
 14. An immunoconjugate comprising the antibody of any one ofclaims 1 to
 13. 15. A nucleotide of a set of nucleotides encoding theantibody of any one of claims 1 to
 13. 16. A vector or a set of vectorscomprising the nucleotide of the set of nucleotides of claim
 15. 17. Ahost cell comprising the nucleotide of the set of nucleotides of claim15 or the vector or the set of vectors of claim
 16. 18. A pharmaceuticalcomposition comprising the antibody of any one of claims 1 to 13, theimmunoconjugate of claim 14, the nucleotide or the set of nucleotides ofclaim 15, the vector or the set of vectors of claim 16, or the host cellof claim 17, and a pharmaceutically acceptable excipient.
 19. A methodof inhibiting a complement pathway in a subject in need thereof,comprising administering to the subject a pharmaceutically effectiveamount of the antibody of any one of claims 1 to 13, the immunoconjugateof claim 14, the nucleotide or the set of nucleotides of claim 15, thevector or the set of vectors of claim 16, or the host cell of claim 17,or the pharmaceutical composition of claim
 18. 20. A method of treatinga complement-mediated disease or disorder in a subject in need thereof,comprising administering to the subject a pharmaceutically effectiveamount of the antibody of any one of claims 1 to 13, the immunoconjugateof claim 14, the nucleotide or the set of nucleotides of claim 15, thevector or the set of vectors of claim 16, or the host cell of claim 17,or the pharmaceutical composition of claim 18.